Composite materials comprising supported porous gels

ABSTRACT

This invention relates to a composite material that comprises a support member that has a plurality of pores extending through the support member and, located in the pores of the support member, and filling the pores of the support member, a macroporous cross-linked gel. The invention also relates to a process for preparing the composite material described above, and to its use. The composite material is suitable, for example, for separation of substances, for example by filtration or adsorption, including chromatography, for use as a support in synthesis or for use as a support for cell growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patentapplication Ser. No. 60/447,730, filed Feb. 19, 2003, entitled“COMPOSITE MATERIALS COMPRISING SUPPORTED POROUS GELS”, the contents ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to composite materials that comprise supportedmacroporous cross-linked gels, and to their preparation and use. Thecomposite materials are suitable, for example, for separation ofsubstances, for example by filtration or adsorption, includingchromatography, for use as a support in synthesis or for use as asupport for cell growth.

BACKGROUND OF THE INVENTION

Composite materials and separation materials have been described inpatent documents such as:

-   U.S. Pat. Nos. 4,224,415; 4,889,632; 4,923,610; 4,952,349;    5,160,627; November 1992; 5,593,576; 5,599,453; 5,672,276;    5,723,601; 5,906,734; 6,045,697; 6,086,769; and 6,258,276;-   International Patent Nos. EP 316,642; WO 00/12618; WO 00/50160; EP    316,642 B1; and EP 664,732 B1; and in other publications, for    example:-   Liu, H. C. and Fried, J. R., Breakthrough of lysozyme through an    affinity membrane of cellulose-Cibaron Blue. AIChE Journal, vol. 40    (1994), p. 40-49. Tennikov, M. B.; Gazdina, N. V.; Tennikova, T. B.;    Svec, F., Effect of porous structure of macroporous polymer supports    on resolution in high-performance membrane chromatography. Journal    of Chromatography A, vol. 798 (1998) p. 55-64.-   Svec, F.; Jelinkova, M.; Votavova, E., Reactive macroporous    membranes based on glycidyl methacrylate-ethylene dimethacrylate    copolymer for high-performance membrane chromatography. Angew.    Makromol. Chem. Vol. 188 (1991) p. 167-176.-   Tennikova, T. B.; Belenkii, B. G.; Svec, F., High-performance    membrane chromatography. A novel method of protein separation. J.    Liquid Chromatography, vol. 13 (1990) p. 63-70.-   Tennikova, T. B.; Bleha, M.; Svec, F.; Almazova, T. V.;    Belenkii, B. G. J., High-performance membrane chromatography of    proteins, a novel method of protein separation. Chromatography, vol.    555 (1991) p. 97-107.-   Tennikova, T. B.; Svec, F. High-performance membrane chromatography:    highly efficient separation method for proteins in ion-exchange,    hydrophobic interaction and reversed-phase modes. J. Chromatography,    vol. 646 (1993) p. 279-288.-   Viklund, C.; Svec, F.; Frechet, J. M. J. Fast ion-exchange HPLC of    proteins using porous poly(glycidyl methacrylate-co-ethylene    dimethacrylate) monoliths grafted with    poly(2-acrylamido-2-methyl-1-propanesulfonic acid). Biotechnol.    Progress, vol. 13 (1997) p. 597-600.-   Mika, A. M. and Childs, R. F. Calculation of the hydrodynamic    permeability of gels and gel-filled microporous membranes, Ind. Eng.    Chem. Res., vol. 40 (2001), p. 1694-1705.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composite material thatcomprises a support member that has a plurality of pores extendingthrough the support member and, located in the pores of the supportmember and essentially filling the pores of the support member, amacroporous cross-linked gel. In some embodiments, the macroporous gelused is responsive to environmental conditions, providing a responsivecomposite material.

In another aspect, the invention provides a process for the separationof substances by means of the composite material described above.

In another aspect, the invention provides a process for solid phasechemical synthesis, wherein the composite material serves as the solidphase in the pores of which the chemical synthesis occurs.

In another aspect, the invention provides a process for growth of amicroorganism or cell, wherein the composite material serves as a solidsupport in the pores of which the growth occurs.

In yet another aspect, the invention provides a process for preparingthe composite material described above, the process comprising:

a) introducing into the pores of the support member a solution orsuspension containing

i) one or more monomers and one or more cross-linking agents that cancombine to form a macroporous gel, or

ii) one or more cross-linkable polymers and one or more cross-linkingagents that can combine to form a macroporous gel,

b) reacting the monomers and the cross-linking agents or the polymersand the cross-linking agents to form a macroporous cross-linked gel thatfills the pores of the support member.

The macroporous gel fills the pores of the support laterally, i.e.substantially perpendicular to the direction of the flow through thecomposite material. By “fill” we mean that, in use, essentially allliquid that passes through the composite material must pass through themacroporous gel. A support member whose pores contain macroporous gel tosuch an amount that this condition is satisfied is regarded as filled.Provided that the condition is met that the liquid passes through themacroporous gel, it is not necessary that the void volume of the supportmember be completely occupied by the macroporous gel.

The porous support member, or host, may be hydrophilic or hydrophobicand can be, for example, in the form of a membrane, a chromatographybed, or a filtration bed. The support member provides the mechanicalstrength to support the macroporous gel. The macroporous gel provides alow resistance to hydraulic flow, enabling high flow rates to beachieved with low reductions in pressure across the composite material.The macroporous gel also provides the separating function of thecomposite material in chromatographic and filtration applications.

A gel is a cross-linked polymer network swollen in a liquid medium. Theswelling liquid prevents the polymer network from collapsing and thenetwork, in turn, retains the liquid.

Gels are typically obtained by polymerization of a monomer and apolyfunctional compound (a cross-linker), or by cross-linking across-linkable polymer, in a solvent which is a good solvent for theformed polymer network and which swells the polymer network. The polymerchains in such a network can be assumed to be uniformly distributedthroughout the whole volume of the network and the average distancebetween the chains, known as mesh size, is determined by thecross-linking density. As the concentration of the cross-linker isincreased, the density of cross-links in the gel also increases, whichleads to a smaller mesh size in the gel. The smaller mesh size resultsin a higher resistance to the flow of liquids through the gel. As theconcentration of the cross-linker is increased further, the constituentsof the gel begin to aggregate, which produces regions of high polymerdensity and regions of low polymer density in the gel. Such gels exhibitwhat has been called microheterogeneity. This aggregation normallycauses the gel to display a higher permeability to liquids, as the flowof liquids takes place primarily through the areas in the gel that havea lower polymer density. The low density areas of the gels are definedas draining regions while the higher density aggregates are callednon-draining regions. As the concentration of the cross-linker isincreased even further, leading to more cross-links, the gel can developregions in which there is essentially no polymer. These regions arereferred to as “macropores” in the present specification.

It is possible to compare the hydrodynamic (Darcy) permeability of aparticular composite material of the invention with a referencematerial. The reference material is obtained by filling the pores of asupport member identical with that of the composite material with ahomogeneous gel of essentially the same chemical composition and thesimilar mass as the gel of the composite material, that is a gelcomposed of the same monomers formed in a good solvent, but cross-linkedonly to such an extent that the gel remains homogeneous and aggregationinto regions of high and low polymer density does not occur. Compositematerials having macroporous gels display hydrodynamic (Darcy)permeabilities that are at least one order of magnitude higher thanthose of the corresponding reference materials, and in some instancesthe permeabilities are more than two or even more than three orders ofmagnitude higher. In this specification, a composite material of theinvention whose hydrodynamic (Darcy) permeability is at least an orderof magnitude greater than that of the corresponding reference materialis said to have a permeability ratio greater than 10.

The permeability ratio is closely related to the size of the macroporesin the composite material. For size-exclusion separations such asultrafiltration, the permeability ratio can be fairly close to 10. Inother applications, for example adsorption, synthesis or cell growth,where larger macropores are used, the permeability ratio can reach, insome embodiments, values of 100 or greater, or even 1000 or greater. Insome instances it is possible to calculate the hydrodynamic permeabilityof homogeneous gels, in accordance with the teachings of Mika A. M. andChilds R. F. Calculation of the hydrodynamic permeability of gels andgel-filled microporous membranes, Ind. Eng. Chem. Res., vol. 40 (2001),p. 1694-1705, incorporated herein by reference. This depends upon datafor the particular gel polymer being available.

From the hydrodynamic permeability there can be derived the hydrodynamicradius, defined as the ratio of the pore volume to the pore wettedsurface area. It can be calculated from the hydrodynamic (Darcy)permeability using the Carman-Kozeny equation as given, for example, inthe book J. Happel and H. Brenner, Low Reynolds Numbers Hydrodynamics,Noordhof of Int. Publ., Leyden, 1973, p. 393, incorporated by referenceherein. It is necessary to assume a value for the Kozeny constant andfor the purpose of these calculations the inventors assume a value of 5.Composite materials of the invention, containing macroporous gels, arefound to have a hydrodynamic radius more than three times as high as thehydrodynamic radius of the corresponding reference material.

From the definition of the hydrodynamic permeability it can be derivedthat two composite materials of the same thickness will havehydrodynamic fluxes at the same pressure that will have the same ratioas their permeability ratio.

The size of macropores in the gel can be within a broad range, from afew nanometers to several hundred nanometers. Preferably, the porous gelconstituent of the composite material has macropores of average sizebetween about 10 and about 3000 nm, has volume porosity between 30 and80% and a thickness equal to that of the porous support member. In someembodiments, the average size of the macropores is preferably between 25and 1500 nm, more preferably between 50 and 1000 nm, and most preferablythe average size of the macropores is about 700 nm.

In the absence of a support member, the macroporous gels used in thepresent invention may be non-self supporting, and they may change oreven lose their porosity when dried. By inserting the macroporous gelwithin a porous support member, mechanical strength is conferred uponthe macroporous gel. The utilization of macroporous gels creates acomposite material that permits larger molecules, such as biologicalmolecules, to enter the macropores and the solution containing suchmolecules to traverse the gel at a high flux.

By a “responsive composite material” is meant a composite material whichcomprises a macroporous gel whose pore-size can be controlled by varyingspecific environmental conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an environmental scanning electron microscope (ESEM) image ofa macroporous poly(APTAC) gel;

FIG. 2 is an ESEM image of a macroporous poly(APTAC) gel incorporatedinto a support member in the form of a membrane;

FIG. 3 is a lysozyme adsorption curve of the membrane prepared inExample 3, below. The membrane volume is 0.467 ml;

FIG. 4 is a BSA adsorption curve of the membrane prepared in Example 8,below;

FIG. 5 is a graphical representation of the hydraulic radius as afunction of mass gain with photo- and thermally initiated porous gelcontaining membranes. Gel: poly(glycidyl methacrylate-co-ethylenediacrylate); solvents: dodecanol(DDC)/cyclohexanol(CHX) 9/91;

FIG. 6 is a graphical representation of the mass gain as a function oftotal monomer concentration during the preparation of compositemembranes;

FIG. 7 is an AFM image of the surface of the AM610 membrane; (scannedarea: 100 μm²)

FIG. 8 shows ESEM images of a nascent (top) and AM610 (bottom) surfaces;(magnification: 5000×);

FIG. 9 shows ESEM images of the surface of AM611 membrane;(magnification: top—5000×, bottom—3500×);

FIG. 10 shows ESEM images of membranes AM610 (top) and AM611 (bottom);(magnification 5000×);

FIG. 11 is a lysozyme adsorption curve of the membrane prepared inExample 15. The membrane volume is 0.501 ml;

FIG. 12 is a lysozyme adsorption curve of the membrane prepared inExample 16. The membrane volume is 0.470 ml;

FIG. 13 is a lysozyme adsorption curve of the membrane prepared inExample 17. The membrane volume is 0.470 ml;

FIG. 14 is an ESEM image of a wet macroporous gel that is the product ofExample 25;

FIG. 15 is an ESEM image of a wet microporous gel in a fibrous non-wovensupport member that is the product of Example 26; and

FIG. 16 shows graphically results of using a multi-membrane stack ofcomposite material of Example 22 in a protein (BSA) adsorption test.

FIG. 17 graphically displays the effect of monomer concentration on themass gain of composite materials.

FIG. 18 graphically displays the effect of ionic interactions on theflux through a composite material at a pressure of 100 kPa.

FIG. 19 graphically displays changes in trans-membrane pressure andpermeate conductivity as a function of salt concentration in thepermeate (A and B) and the changes of trans-membrane pressure as afunction of permeate conductivity (salt concentration) (C).

FIG. 20 shows the relationship between trans-membrane pressure,conductivity and absorbance for the HIgG ultrafiltration carried out inExample 39.

FIG. 21 shows the relationship between conductivity and absorbance forthe HSA ultrafiltration carried out in Example 39.

FIG. 22 shows the relationship between trans-membrane pressure,conductivity and absorbance for the HSA/HIgG ultrafiltration carried outin Example 39.

DETAILED DESCRIPTION OF THE INVENTION General Characteristics

Preferably, the macroporous gel is anchored within the support member.The term “anchored” is intended to mean that the gel is held within thepores of the support member, but the term is not necessarily restrictedto mean that the gel is chemically bound to the pores of the supportmember. The gel can be held by the physical constraint imposed upon itby enmeshing and intertwining with structural elements of the host,without actually being chemically grafted to the host or support member,although in some embodiments, the macroporous gel may become grafted tothe surface of the pores of the support member.

It will be appreciated that as the macropores are present in the gelthat fills the pores of the support member, the macropores must besmaller than the pores of the support member. Consequently, the flowcharacteristics and separation characteristics of the composite materialare dependent on the characteristics of the macroporous gel, but arelargely independent of the characteristics of the porous support member,with the proviso, of course, that the size of the pores present in thesupport member is greater than the size of the macropores of the gel.The porosity of the composite material can be tailored by filling thesupport member with a gel whose porosity is primarily controlled by thenature and amounts of monomer or polymer, cross-linking agent, reactionsolvent, and porogen, if used. As pores of the support member are filledwith the same macroporous gel material, there is achieved a high degreeof consistency in properties of the composite material, and for aparticular support member these properties are determined largely, ifnot entirely, by the properties of the macroporous gel. The net resultis that the invention provides control over macropore-size, permeabilityand surface area of the composite materials.

The number of macropores in the composite material is not dictated bythe number of pores in the support material. The number of macropores inthe composite material can be much greater than the number of pores inthe support member, although the macropores are smaller than the poresin the support member. As mentioned above, the effect of the pore-sizeof the support material on the pore-size of the macroporous gel isgenerally quite negligible. An exception to this is found in those caseswhere the support member has a large difference in pore-size andpore-size distribution, and where a macroporous gel having very smallpore-sizes and a narrow range in pore-size distribution is sought. Inthese cases, large variations in the pore-size distribution of thesupport member are weakly reflected in the pore-size distribution of themacroporous gel. As such it is preferable to use a support member with asomewhat narrow pore-size range in these situations.

The properties of the composite materials can be tuned, by adjusting theaverage pore diameter of the macroporous gel. For some purposes, forexample ultrafiltration by means of size exclusion, small pores may berequired. For other purposes, for example use as a solid support for achemical synthesis involving fast-kinetics, large pores may be required.The size of the macropores is mainly dependent on the nature andconcentration of the cross-linking agent, the nature or the solvent orsolvents in which the gel is formed, the amount of any polymerizationinitiator or catalyst and, if present, the nature and concentration ofporogen.

Generally, as the concentration of cross-linking agent is increased, thesize of the macropores in the gel is also increased. For example, themolar ratio of polyfunctional compound(s) (cross-linking agent) tomonomer(s) may be in the range of from about 5:95 to about 70:30,preferably in the range of from about 10:90 to about 50:50, and morepreferably in the range of from about 15:85 to about 45:55.

The components of the macroporous gel are introduced into the pores ofthe support member by means of a liquid vehicle, and solvent selectionfor in situ polymerization or cross-linking plays a role in obtainingporous gels. Generally, the solvent or solvent mixture should dissolvemonomers and polyfunctional compounds, or cross-linkable polymers andcross-linking agents, over a wide range of concentrations. If thesolvent is a good solvent for the gel polymer, porosity can only beintroduced into the gel by cross-linking or porogen. If, however, thereis present a solvent that is a thermodynamically poor solvent ornon-solvent, this solvent will act as a porogen. By combining solventsof different affinities to the gel polymer, from a good solvent througha poor solvent to a non-solvent, at different ratios, both porosity andpore dimensions can be altered. In general, the poorer the solvent orthe solvent mixture the higher the porosity and the sizes of macropores.Preferably, the solvent or solvent mixture for in situ polymerizationcontains poor solvent in the range from about 0% to about 100%, morepreferably from about 10% to about 90%. Examples of good solvents forpoly(2-acrylamido-2-methyl-1-propanesulfonic acid) are water andN,N-dimethylformamide. Examples of poor solvents include dioxane,hydrocarbons, esters, and ketones. An example of a good solvent forpoly(acrylamide) is water. Examples of poor solvents include dioxane,alcohols such as methanol, N,N-dimethylformamide, hydrocarbons, esters,and ketones. Preferably, the solvents used are miscible with water.

When the polymerization is carried out using a liquid vehicle thatcontains non-solvents or poor solvents, the resulting structure is oftenbuilt of clusters of agglomerated microspheres that form the body of themacroporous gel. The pores in such materials consist of the voidslocated between clusters (macropores), voids between microspheres in theclusters (mesopores), and pores inside the microspheres themselves(micropores).

Porogens can be broadly described as pore generating additives. Examplesof porogens that can be used in the gel-forming reaction includethermodynamically poor solvents or extractable polymers, for examplepoly(ethyleneglycol), or surfactants, or salts. Porogens are known inthe art, and a person skilled can determine, using standard experimentaltechniques and without exercise of any inventive faculty, which porogensare suitable to prepare macroporous gels for use in a desired compositematerial.

There is no simple way to predict accurately the structure parameters ofporous gels obtained under given conditions, but qualitative rules areavailable to give some guidance. Generally, the mechanism of porous gelformation via polymerization of one or more monomers and cross-linkersinvolves, as a first step, an agglomeration of polymer chains to givenuclei. The polymerization continues both in the nuclei and in theremaining solution to form microspheres which grow in size by capturingnewly precipitated nuclei and polymers from the solution. At some point,the microspheres become interconnected with each other in large clustersthat form the body of the macroporous gel. The poorer the solventquality the faster nucleation occurs during the gel-forming process. Ifthe number of nuclei formed is very large, as in the case of highconcentration of a polymerization initiator, smaller pores may beexpected. If, however, the number of nuclei is smaller and the reactionkinetics is such that the nuclei can grow larger, large pores are formedin the gel. High concentration of a cross-linker usually causes earlynucleation. The nuclei, however, may be too highly cross-linked to beable to swell with the monomers, grow and coalesce in clusters. This mayresult in very small pores. Because of the different ways that thepolymerization may proceed and the polymerization conditions may affectthe gel porous structure, a large variety of structures can be obtainedbut conditions for each of the structures need to be determinedexperimentally.

Separations with the Composite Material

In some embodiments of the invention the composite material is used as aseparating medium, for example in filtration operations where a liquidto be filtered is passed through the composite membrane and separationof one or more components from the liquid is effected by size exclusionin an uncharged macroporous gel. The separation can further be enhancedby the Donnan exclusion of charged molecules by use of a chargedmacroporous gel. If the macroporous gel contains a fixed charge and thecharge of the solutes can be appropriately adjusted, the solutes can beseparated even against their size gradient. For example with a solutioncontaining a mixture of proteins, if a pH value is selected for whichone of the proteins in the mixture is at its isoelectric point while theother proteins retain charge of the same sign as the membrane charge,the other proteins can be held back in the retentate because of thecharge repulsion with the membrane. By tailoring the conditions forfractionation, good selectivity, even for proteins of the same size, canbe obtained.

Separation can also be achieved by the presence of reactive functionalgroups in the macroporous gel. These functional groups can be used tobear a ligand or other specific binding site that has an affinity to amolecule or ion, including a biomolecule or biomolecular ion. When aliquid containing the particular molecule or ion is passed through thecomposite material the ligand or specific binding site interacts withthe molecule or ion enough to adsorb it. In some cases it is possible tosubsequently desorb the captured molecule or ion when the environmentaround the composite material is subsequently altered, for example bychanging the nature of the solvent passed through the macropores of thegel. The binding sites can also include charged groups.

Composition of the Macroporous Gels

The macroporous gels can be formed through the in-situ reaction of oneor more polymerisable monomers with one or more cross-linkers, or of oneor more cross-linkable polymers with one or more cross-linker to form across-linked gel that has macropores of a suitable size. Suitablepolymerisable monomers include monomers containing vinyl or acrylgroups. For Donnan exclusion, there can be used vinyl or acryl monomerscontaining at least one polar and/or ionic functional group, orfunctional group that can be converted into ionic group. For biologicalaffinity there can be used vinyl or acryl monomers containing at leastone reactive functional group. Preferred polymerisable monomers includeacrylamide, 2-acryloxyethyltrimethylammonium chloride,N-acryloxysuccinimide, N-acryloyltris(hydroxymethyl)methylamine,2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, butyl acrylate and methacrylate, N,N-diethylacrylamide,N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate andmethacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide,N,N-dimethylacrylamide, n-dodecyl acrylate, n-dodecyl methacrylate,dodecyl methacrylamide, ethyl methacrylate, 2-(2-ethoxyethoxy)ethylacrylate and methacrylate, 2,3-dihydroxypropyl acrylate andmethacrylate, glycidyl acrylate and methacrylate, n-heptyl acrylate andmethacrylate, 1-hexadecyl acrylate and methacrylate, 2-hydroxyethylacrylate and methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl acrylate and methacrylate, methacrylamide, methacrylicanhydride, methacryloxyethyltrimethylammonium chloride,2-(2-methoxy)ethyl acrylate and methacrylate, octadecyl acrylamide,octylacrylamide, octyl methacrylate, propyl acrylate and methacrylate,N-iso-propylacrylamide, stearyl acrylate, styrene, 4-vinylpyridine,vinylsulfonic acid, N-vinyl-2-pyrrodinone. Particularly preferredmonomers include dimethyldiallylammonium chloride,acrylamido-2-methyl-1-propanesulfonic acid (AMPS), (3-acrylamidopropyl)trimethylammonium chloride (APTAC), acrylamide, methacrylic acid (MAA),acrylic acid (AA), 4-styrenesulfonic acid and its salts, acrylamide,glycidyl methacrylate, diallylamine, and diallylammonium chloride.

The crosslinker may be, for example, a compound containing at least twovinyl or acryl groups. Examples of crosslinkers includebisacrylamidoacetic acid, 2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate anddimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanedioldiacrylate and dimethacrylate, 1,10-dodecanediol diacrylate anddimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,2,2-dimethylpropanediol diacrylate and dimethacrylate, dipentaerythritolpentaacrylate, dipropylene glycol diacrylate and dimethacrylate,N,N-dodecamethylenebisacrylamide, divinylbenzene, glyceroltrimethacrylate, glycerol tris(acryloxypropyl)ether,N,N′-hexamethylenebisacrylamide, N,N′-octamethylenebisacrylamide,1,5-pentanediol diacrylate and dimethacrylate, 1,3-phenylenediacrylate,poly(ethylene glycol) diacrylate and dimethacrylate, poly(propylene)diacrylate and dimethacrylate, triethylene glycol diacrylate anddimethacrylate, triethylene glycol divinyl ether, tripropylene glycoldiacrylate or dimethacrylate, diallyl diglycol carbonate, poly(ethyleneglycol) divinyl ether, N,N′-dimethacryloylpiperazine, divinyl glycol,ethylene glycol diacrylate, ethylene glycol dimethacrylate,N,N′-methylenebisacrylamide, 1,1,1-trimethylolethane trimethacrylate,1,1,1-trimethylolpropane triacrylate, 1,1,1-trimethylolpropanetrimethacrylate, vinyl acrylate, 1,6-hexanediol diacrylate anddimethacrylate, 1,3-butylene glycol diacrylate and dimethacrylate,alkoxylated cyclohexane dimethanol dicarylate, alkoxylated hexanedioldiacrylate, alkoxylated neopentyl glycol diacrylate, aromaticdimethacrylate, caprolacone modified neopentylglycol hydroxypivalatediacrylate, cyclohexane dimethanol diacrylate and dimethacrylate,ethoxylated bisphenol diacrylate and dimethacrylate, neopentyl glycoldiacrylate and dimethacrylate, ethoxylated trimethylolpropanetriarylate, propoxylated trimethylolpropane triacrylate, propoxylatedglyceryl triacrylate, pentaerythritol triacrylate, tris (2-hydroxyethyl)isocyanurate triacrylate, di-trimethylolpropane tetraacrylate,dipentaerythritol pentaacrylate, ethoxylated pentaerythritoltetraacrylate, pentaacrylate ester, pentaerythritol tetraacrylate, andcaprolactone modified dipentaerythritol hexaacrylate. Particularlypreferred cross-linking agents include N,N′-methylenebisacrylamide,diethylene glycol diacrylate and dimethacrylate, trimethylolpropanetriacrylate, ethylene glycol diacrylate and dimethacrylate,tetra(ethylene glycol) diacrylate, 1,6-hexanediol diacrylate,divinylbenzene, poly(ethylene glycol) diacrylate.

The concentration of monomer in the macroporous gel can have an effecton the resiliency of the macroporous gel prepared. A low monomerconcentration can lead to a macroporous gel that is non-self supporting.Such non-self supporting gels might be advantageous as adsorbents, asthey could lead to gels having greater adsorption capacity. In someembodiments, the monomer concentration is 60% or less, for example about60, 50, 40, 30, 20, 10 or 5%.

When a cross-linkable polymer is used, it can be dissolved and reactedin-situ in the support with a cross-linking agent to form themacroporous gel. Suitable cross-linkable polymers includepoly(ethyleneimine), poly(4-vinylpyridine), poly(vinylbenzyl chloride),poly(diallylammonium chloride), poly(glycidyl methacrylate),poly(allylamine), copolymers of vinylpyridine anddimethyldiallylammonium chloride, copolymers of vinylpyridine,dimethyladiallylammonium chloride, or(3-acrylamidopropyl)trimethylammonium chloride with glycidyl acrylate ormethacrylate, of which poly(ethyleneimine), poly(diallylammoniumchloride), and poly(glycidyl methacrylate) are preferred. Use ofcross-linkable polymers instead of monomers can, in some instances,require a decrease in the concentration of cross-linking agent. In orderto retain the large size of the pores in the gel with a lowercross-linking agent concentration, a porogen can be added to the mixtureused to prepare the macroporous gel.

The cross-linking agent for reaction with the cross-linkable polymer isselected from molecules containing two or more reactive groups that canreact with an atom or group of atoms in the polymer to be cross-linked,such as epoxy groups or alkyl/aryl halides that can react with nitrogenatoms of polyamines, or amine groups that can react with alkyl/arylhalides or epoxy groups of glycidyl-group-containing polymers to be insitu cross-linked. Suitable cross-linkers include ethylene glycoldiglycidyl ether, poly(propylene glycol) diglycidyl ether,1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane,1,6-dibromohexane, α,α′-dibromo-p-xylene, α,α′-dichloro-p-xylene,1,4-dibromo-2-butene, piperazine, 1,4-diazabicyclo[2.2.2]octane,1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane,1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane.

It is also possible to modify polymers containing reactive groups suchas an amino, hydroxyl, carboxylic acid, carboxylic acid ester, or epoxygroups with reagents to introduce vinyl groups that can be subsequentlypolymerized by treatment with a polymerization initiator to form amacroporous gel. Examples of suitable vinyl groups that can beintroduced include vinylbenzene derivatives, allyl derivatives, acrolyland methacrolyl derivatives. The cross-linking of these vinylsubstituted polymers can in some instances be facilitated by theintroduction of further monomers such as acrylamide, N-vinylpyrrolidone,acrylic and methacrylic acids and their salts.

Macromonomers can also be used as monomers or as cross-linking agents.Macromonomers can be polymers or oligomers that have one(monofunctional) or more (cross-linking agent) reactive groups, often atthe ends, which enable them to act as a monomer or a cross-linker. Formonomers, each macromonomer molecule is attached to the main chain ofthe final polymer by reaction of only one monomeric unit in themacromonomer molecule. Examples of macromonomers include poly(ethyleneglycol)acrylate and poly(ethylene glycol)methacrylate, while examples ofpolyfunctional macromonomers include poly(ethylene glycol)diacrylate andpoly(ethylene glycol)dimethacrylate. Macromonomers preferably havemolecular weights of about 200 Da or greater.

Many macroporous gels can be prepared, including neutral hydrogels,charged hydrogels, polyelectrolyte gels, hydrophobic gels, and neutraland functional gels.

If the gel selected is a neutral hydrogel or a charged hydrogel forwhich water is the swelling liquid medium, the resulting supportedmacroporous gel is normally quite hydrophilic. Hydrophilic compositematerials are preferred as they provide better flow characteristics andimpart anti-fouling tendencies to the membranes. Examples of suitablehydrogels include cross-linked gels of poly(vinyl alcohol),poly(acrylamide), poly(isopropylacrylamide), poly(vinylpyrrolidone),poly(hydroxymethyl acrylate), poly(ethylene oxide), copolymers ofacrylic acid or methacrylic acid with acrylamide, isopropylacrylamide,or vinylpyrrolidone, copolymers of acrylamide-2-methyl-1-propanesulfonicacid with acrylamide, isopropylacrylamide, or vinylpyrrolidone,copolymers of (3-acrylamidopropyl) trimethylammonium chloride withacrylamide, isopropylacrylamide, or N-vinylpyrrolidone, copolymers ofdiallyldimethylammonium chloride with acrylamide, isopropylacrylamide,or vinylpyrrolidone. Preferred hydrogels include cross-linked poly(vinylalcohol), poly(acrylamide), poly(isopropylacrylamide) andpoly(vinylpyrrolidone) and cross-linked copolymers of neutral monomerssuch as acrylamide or N-vinylpyrrolidone with charged monomers such asacrylamide-2-methyl-1-propanesulfonic acid or diallyldimethylammoniumchloride.

The macroporous gels can be selected to comprise polyelectrolytes. Likethe charged hydrogels, polyelectrolyte gels give hydrophilic compositematerial, and they also carry a charge. The polyelectrolyte gel can beselected, for example, from cross-linkedpoly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts,poly(styrenesulfonic acid) and its salts, poly(vinylsulfonic acid) andits salts, poly(alginic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium)salts, poly(vinylbenzyl-N-trimethylammonium) salts, poly(ethyleneimine)and its salts. Preferred charged gels include cross-linked poly(acrylicacid) and its salts, poly(methacrylic acid) and its salts,poly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, and poly(4-vinylpyridinium) salts.

One of the differences between charged gels and polyelectrolyte gels isthat the repeating monomer in the polyelectrolyte gel bears a charge,while in the charged gel, the charge is found in a co-polymerized unitthat is randomly distributed through the polymer. The monomer used toform the polyelectrolyte gel or the co-polymer in the charged gel thatbears a charge usually has a charge bearing group, but it can also be anon-charge-bearing group that can become charged in a post-gelationprocess (e.g. quaternization of nitrogen bearing groups). Examples ofpolymers that can become charged include poly(4-vinylpyridine) which canbe quaternized with various alkyl and alkylaryl halides. Suitable alkylhalides include those having up to 8 carbon atoms, for example methyliodide, ethyl bromide, butyl bromide, and propyl bromide. Suitablealkylaryl halides include benzyl halides, especially benzyl chloride andbenzyl bromide. Another polymer that can become charged ispoly(vinylbenzyl chloride), which can be quaternized with variousamines, for example, lower alkylamines or aromatic amines such astriethylamine, pyridine, azabicyclo[2.2.2]octane, N-methylpyrrolidine,and N-methylpiperidine, and lower hydroxyalkylamines, for exampletriethanolamine. Yet another polymer that can become charged ispoly(glycidyl methacrylate) or poly(glycidyl acrylate), which can reactwith various amines, for example lower alkylamines such as diethylamineand triethylamine, azabicyclo[2.2.2]octane, N-methylpyrrolidine, andN-methylpiperidine. Alternatively, glycidyl moieties can be converted tosulfonic acid groups by reaction with, for example alkali metal sulfitessuch as sodium sulfite. A person skilled in the art will appreciate thatthere are other polymers that are, or can be rendered, charge-bearing.

The macroporous gel can be selected to comprise hydrophobic monomers topermit separations in organic solvents, for example hydrocarbons,especially liquid paraffins such as hexanes. Hydrophobic monomers, suchas styrene and its derivatives, for example an alkyl substituted styrenederivative such as para-tertbutyl styrene, can be used to preparehydrophobic macroporous gels. Copolymers of these monomers can be used.

A macroporous gel comprising hydrophobic monomers can be used to capturemolecules from fluids passing through the pores by hydrophobicinteractions.

As stated above, the macroporous gels can also be selected to comprisereactive functional groups that can be used to attach ligands or otherspecific binding sites. These functional macroporous gels can beprepared from cross-linked polymers bearing functional groups, forexample epoxy, anhydride, azide, reactive halogen, or acid chloridegroups, that can be used to attach the ligands or other specific bindingsites. Examples include cross-linked poly(glycidyl methacrylate),poly(acrylamidoxime), poly(acrylic anhydride), poly(azelaic anhydride),poly(maleic anhydride), poly(hydrazide), poly(acryloyl chloride),poly(2-bromoethyl methacrylate), poly(vinyl methyl ketone).Functionality that can be introduced can take the form of antibodies orfragments of antibodies, or alternatively, chemical mimics such as dyes.Functional gels are attractive in biomolecule purifications orseparations, as they can offer preferential binding to certain moleculesby binding to active sites, while being non-reactive to other molecules,even when there is no significant difference in size between themolecules, examples being affinity ligands selected to bind with someproteins but not others. Affinity ligands that can be attached to porousgels via reactive groups include amino acid ligands such asL-phenylalanine, tryptophan, or L-histidine to separate γ-globulins andimmunoglobulins, antigen and antibody ligands such as monoclonalantibodies, protein A, recombinant protein A, protein G, or recombinantprotein G to separate immunoglobulins from different media, dye ligandssuch as cibaron blue or active red to separate albumins and variousenzymes, metal affinity ligands such as complexes of iminodiacetic acid(IDA) ligand with Cu²⁺, Ni²⁺, Zn²⁺, or Co²⁺ to separate various proteinssuch as histidine, lysozyme, or tryptophan from various media.

Responsive Macroporous Gels

Polymers that change their conformation in response to changes inenvironmental conditions are known. By incorporating the properties ofsuch polymers in the macroporous gel, a composite material with dynamicpore-size is obtained. These composite materials having responsivecharacteristics are substantially the same as the composite materialsdescribed above, except that at least one of the monomers or polymersthat form the macroporous gel has a chemical structure which facilitateschanges in pore-size.

The changes in the pore-size of the macroporous gel are due to thephysical relationship between the support member and the macroporousgel. The composite material can be described as having three distinctzones: (a) the support member, which ideally does not change shape, (b)the incorporated macroporous gel that “fills” the pores of the supportmember, and (c) the volume within the macropores of the gel, whichvolume is filled with water or a solvent and in which is found verylittle or no gel polymer. Under pressure, hydraulic flow occurs throughthe macropores of the gel, and the flux through the composite materialis related to the number of pores in the macroporous gel, the radius ofthese pores, and the tortuosity of the path of the pores in themacroporous gel through the composite material.

As the degree of swelling of the macroporous gel is changed by anenvironmental stimulus, the total volume occupied by the macroporous gelis constrained by the fixed total volume defined by the support member.As the overall volume of the macroporous gel is constrained by thesupport member, by necessity the volume fraction of the gel expands intothe area defined by macropores in the gel. As the number of macroporesand their tortuosity remain essentially constant with the change involume fraction of the macroporous gel, the diameter or radius of themacropores themselves must change. If the macroporous or structured gelwere unconfined, the environmentally induced changes would cause thetotal volume of the swollen gel to change. As such it would not followin this unconfined case that the changes would result in a controllablechange in pore-size of the macroporous gel.

The reason behind the change in volume of the macroporous gel is relatedto interactions between the polymer structures that form the gels, orthe interactions between the polymer chains and the solvents or solutespresent in the solvent that diffuse into the gel. The changes in thevolume occupied by the gel are linked to the conformation adopted by thepolymer chains that form the macroporous gels. The natural tendency ofthe polymer chains is to coil around themselves, which leads to a gelhaving a smaller volume. If the polymer chains within the gel can bemanipulated to uncoil and form a more rigid backbone, the overall volumeof the gel will increase. It is thus this coiling/uncoiling which isaffected by the environmental stimuli that are applied to the responsivecomposite material.

The volume changes of the pores can either be “continuous” or“discontinuous”. A continuous volume change takes place over arelatively large change in the triggering environmental condition andwhere there exists at least one stable volume near the transitionbetween the swollen and collapsed state. Preferably, a continuous volumechange will go through a multitude of stable transition volumes betweenthe swollen and the collapsed state. A discontinuous volume change ingels is characterised by the reversible transition from swollen tocollapsed state taking place over an extremely small change in thetriggering environmental condition, for example, less than 0.1 pH unitor 0.1 degree Celsius. Gels exhibiting discontinuous volume change arecalled “phase-transition” gels and systems or devices with such gels areoften called “chemical valves”. Preferably, the responsive macroporousgels according to this embodiment of the invention undergo a“continuous” volume change through discrete stable volumes that can beutilized to control the pore-size of the gel.

Of the environmental stimuli that can be used to change the pore-size inthe responsive macroporous, mention is made of pH, specific ions, ionicstrength, temperature, light, electric fields, and magnetic fields. Theeffect of each stimulus, and examples of monomers that react to such astimulus, will be described in more detail below.

One stimulus that can be utilised to change the pore-size of responsivemacroporous gel is the pH of the solution being passed through the poresof the gel. A change in the pH of the solution will affect the pore-sizeof the gel if the gel comprises weak acids or weak bases. In such cases,the natural tendency of the polymer chain within the gel to coil arounditself will be balanced by the repulsion between the charged groups(weak acidic or basic groups) along the length of the polymer chain.Variations in the amount of charge along the chain cause large changesin conformation of the polymer chain, which in turn causes changes inthe volume occupied by the gel. Changes in the pH of the solution areeffective at controlling the amount of repulsion along the polymerchain, as they change the degree of ionisation of the charged groups. Agel comprising weak acid groups becomes less ionised as the pH islowered and the gel contracts. Conversely, a weak base becomes moreionised as the pH is lowered and the chain elongates or stretches togive a swollen gel.

Examples of monomers that have weak acid functionality include acrylicacid, methacrylic acid, itaconic acid, 4-vinylbenzoic acid,bisacrylamidoacetic acid, and bis(2-methacryloxyethyl) phosphate.Examples of monomers that have weak base functionality include2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, 2-(tert-butylamino)ethyl methacrylate, diallylamine,2-(N,N-diethylamino)ethyl methacrylate, 2-(N,N-dimethylamino)ethylacrylate, 2-(N,N-dimethylamino)ethyl methacrylate, 1-vinylimidazole, and4-vinylpyridine. Glycidyl methacrylate derivatizedhyaluronate-hydroxyethyl acrylate based hydrogels can also be used toprepare composite materials that are pH responsive [Inukai M., Jin Y.,Yomota C., Yonese M., Chem. Pharm. Bull., (2000), 48:850-854; which ishereby incorporated by reference].

Variations in pH have little effect on the degree of ionisation ofstrong acids and bases, and as such, only drastic variations in pH caneffect pore-size changes in gels comprising these functionalities.

Another stimulus that can be utilised for changing the pore-size of aresponsive macroporous gel is the salt concentration of the solutionbeing passed through the pores of the gel. Similarly to variations inpH, variations in salt concentration will effect pore-size variations inmacroporous gels that comprise weak acidic or weak basic groups. Thereason for the changes in pore-size, however, does differ slightly. Theaddition of an ionic solute has the ability to shield the charged groupsfound on the polymer chain in the gel by the formation of ion-pairs.This lessens the coulombic repulsion between the adjacent chargedgroups, which allows the chain to relax into a coiled conformation. Anincrease in salt concentration will shield both a weak acid group and aweak base group. Therefore, when the salt concentration is increased,for example by adding a concentrated salt solution to the bulk solutionbeing passed through the composite material, the shielding effect of theadditional ions leads to an increase in pore size. Alternatively, adecrease in salt concentration, such as obtained by diluting the bulksolution being passed through the composite material, will lead to lessshielding and a smaller pore size.

Changes in salt concentration can also be used with macroporous gelsthat comprise strong acid groups and strong basic groups, as thesegroups are also shielded by the presence of free ionic species.

Examples of monomers that bear weak acid or base groups are listedabove. Examples of monomers that have strong acid functionality include2-acrylamido-2-methyl-1-propanesulfonic acid, sodium2-methyl-2-propene-1-sulfonate, sodium styrenesulfonate, and sodiumvinylsulfonate. Examples of monomers that have strong basicfunctionality include 2-acryloxyethyltrimethylammonium chloride,diallyldimethylammonium chloride, methacrylamidopropyltrimethylammoniumchloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.Ionic functionality (weak/strong acids and bases) may also be introducedin macroporous gels that do not originally bear charged functionalitiesbut that bear instead reactive groups that can be converted into ionicor ionisable moieties in a post-polymerization treatment. Suitablemonomers with reactive groups include acrylic anhydride, allyl glycidylether, bromostyrene, chloromethylstyrene, chlorostyrenes, glycidylacrylate, glycidyl methacrylate, 4-hydroxybutyl methacrylate,2-hydroxyethyl acrylate, methacryloyl chloride. For example, amacroporous gel comprising a glycidyl acrylate or methacrylate group canbe treated with diethylamine to introduce weak base functionality orwith sodium sulfite in an iso-propanol/water mixture to introduce strongacid (sulfonic acid) functionality.

Another stimulus that can be used to change the pore-size of aresponsive macroporous gel is the temperature of the gel. Variousmethods are available for changing the temperature of the macroporousgel, one of which includes changing the temperature of a liquid flowingthrough the pores of the macroporous gel. While the change in overallgel volume for temperature dependant gels is again due to the control ofthe coiling or uncoiling of the polymer chains that form the gel, thecontraction or expansion of the gel is not linked to the presence ofcharged groups on the polymer chain. For temperature dependant gels, theamount solvation of the polymer chain controls the conformation of thepolymer chain. At lower temperatures the chains are solvated, whichallows an elongated conformation of the polymer chain. As thetemperature is increased, an entropic desolvation takes place causingthe chains to coil and contract. Therefore, increases in temperaturelead to larger pore sizes in the gel while decreases in temperature leadto smaller pore sizes.

Macroporous gels that comprise hydrophobic monomers are most suitablefor use in temperature dependant systems, as solvation effects aremarkedly observed for polymers that have hydrophobic functionality.Examples of monomers that have hydrophobic functionality includeN-(propyl)acrylamide, N-(tert-butyl)acrylamide, butyl acrylates, decylacrylates, decyl methacrylates, 2-ethylbutyl methacrylate, n-heptylacrylate, n-heptyl methacrylate, 1-hexadecyl acrylate, 1-hexadecylmethacrylate, n-hexyl acrylate, n-hexyl methacrylate, andN-(n-octadecyl)acrylamide. Gels displaying thermal response can also beprepared from sulphated hyaluronic acid-based gels (see Barbucci R.,Rappuoli R., Borzacchiello A., Ambrosio L., J. Biomater. Sci.-Polym.Ed., (2000), 11:383-399), incorporated herein by reference.

Light is another stimulus that can be used to change the pore-size ofthe responsive macroporous gel. Light induced changes are due tophotoisomerizations in the backbone or the side-chains of the polymerchains that form the gel. These photoisomerizations cause a change ineither the conformation and local dipole moment, or in the degree ofionisation through light induced electron transfer reactions. One typeof monomer that is suitable for use in light controlled systemscomprises unsaturated functionalities than can undergo a trans-cisisomerization on irradiation. Examples of photoresponsive monomers thatgo through cis-trans conformation and dipole changes include4-(4-oxy-4′-cyanoazobenzene)but-1-yl methacrylate,6-(4-oxy-4′-cyanoazobenzene)hex-1-yl methacrylate,8-(4-oxy-4′-cyanoazobenzene)oct-1-yl methacrylate,4-[ω-methacryloyloxyoligo(ethyleneglycol)]-4′-cyanoazobenzene,4-methacryloyloxy-4′-{2-cyano-3-oxy-3-[ω-methoxyoligo(ethyleneglycol)]prop-1-en-1-yl}azobenzene,and methacrylate monomers containing a mesogenic group and aphotochromic para-nitroazobenzene group. It is also possible toincorporate the photoresponsive moeity in the crosslinker instead of themonomer. Examples of photoresponsive crosslinkers that go throughcis-trans conformation and dipole changes include4,4′-Divinylazobenzene,N,N′-bis(β-styrylsulfonyl)-4,4′-diaminoazobenzene,4,4′-bis(methacryloylamino)azobenzene, 4,4′-dimethacryloylazobenzene,and bis((methacryloyloxy)methyl)spirobenzopyran.

The pore-size of the gel can also be altered by subjecting themacroporous gel to an electric field or to an electrical current. Theresponse of the gel to electrochemical current changes is closelyrelated to the pH systems described above. This close relationship isdue to the fact that the passage of an electrochemical current throughan aqueous system causes a “water splitting” reaction, which reactionleads to changes in the pH of the aqueous system. Electrical current canbe passed through a composite material of the invention e.g. by placingan electrode at either end of the composite material. When currentdifferential is applied to the electrodes, water molecules will separateand concentrations of H⁺ and HO⁻ will increase at their respectiveelectrodes. As described earlier, changes in pH can be used to controlthe pore-size of macroporous gels that comprise weak acid or weak basefunctionalities, which control is linked to the relationship between theionisation of these functionalities and the coiling/uncoiling of thepolymer chains that form the gel. Examples of weak acidic and weak basicmonomers are given above.

Changes in gel volume due to fluctuations in an electrical field havebeen previously observed, such as in Murdan S., J. Control. Release,(2003), 92:1-17; and in Jensen M., Hansen P. B., Murdan S., Frokjaer S.,Florence A. T., Eur. J. Pharm. Sci., (2002), 15:139-148, which arehereby incorporated by reference. While the exact process through whichthe gel volume is changed by the application of an electrical field isnot yet well defined, the volume change itself is well documented.Chondroitin 4-sulphate (CS) is an example of a monomer that isresponsive to electrical field fluctuations.

In some embodiments, the various stimuli response systems can becombined to offer gels that respond to more than one stimulus. Anexample of such a combined system can be prepared by combining a chargedpolymer (weak/strong acid or base) with a hydrophobic monomer. Themacroporous gel resulting from such a combination will display responsesto changes in salt concentration, changes in solution pH (when weakacids or bases are used), and changes in temperature. When combiningdifferent monomers, it is possible that the responsiveness of the gel toa single of the stimuli will be diminished, as the concentration of themonomer that responds to that particular stimulus will be lowered in thegel.

The magnitude of the response expressed by the macroporous gels, whenvarious stimuli are applied to the gel, depends many differentvariables, a few of which are discussed below:

The responsiveness of the macroporous gel is dependent on theconcentration of the crosslinking agent. Generally, as the concentrationof cross-linking agent is increased, the size of the macropores in theresponsive gel is also increased, but the range of pore-size changes isdecreased. This relationship is fairly straightforward, as a higherconcentration of crosslinks within the gel will limit the amount ofcoiling and uncoiling that will be available to the responsive gel. Themolar ratio of crosslinking agent(s) to monomer(s) may be in the rangeof from about 5:95 to about 40:60, preferably in the range of from about7.5:92.5 to about 10:90, and more preferably in the range of from about10:90 to about 25:75.

Certain stimuli naturally evoke a broader range of response in the gel,as they more effectively affect the conformation of the polymer chainsthat form the gel. For example, variations in pH or temperature evoke astrong response from the appropriate macroporous gels, while changes insalt concentrations and light intensity evoke a slightly smallerresponse.

The concentration of the responsive monomer in the gel also affects thelevel of response demonstrated by the gel. Preferably, the responsivemacroporous gels are composed of one or more responsive monomers and ofone or more neutral monomers. The presence of a neutral monomer isimportant in those systems that have a very strong response to changesin the environmental conditions, as such systems often displaydiscontinuous responses in pore-size (valve-effects). Addition of aneutral monomer attenuates the response, permitting a more controlledchange in pore-size. Preferably, the molar ratio of the neutral monomersto the molar ratio of responsive monomers in the responsive macroporousgel is in the range from 5:95 to 95:5, more preferably in the range from25:75 to 75:25, and more preferably in the range from 40:60 to 60:40.Suitable neutral monomers include acrylamide, N-acryloylmorpholine,N-acryloxysuccinimide, 2-acrylamido-2-(hydroxymethyl)-1,3-propanediol,N,N-diethylacrylamide, N,N-dimethylacrylamide, 2-(2-ethoxyethoxy)ethylacrylate, 2-ethoxyethyl methacrylate, 2,3-dihydroxypropyl methacrylate,2-hydroxyethyl methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl methacrylate, methacrylamide, N-[tris(hydroxymethyl)methyl]-1-methacrylamide, N-methylmethacrylamide,N-methyl-N-vinylacetamide, poly(ethylene glycol) monomethacrylate,N-iso-propylacrylamide, N-vinyl-2-pyrrolidone.

Porous Support Member

A variety of materials can be used to form the support member; however,apart from materials such as cellulose and some of its derivatives, mostof these materials are strongly or relatively hydrophobic. Hydrophobicfiltration membranes are not usually desired for use with aqueoussystems, as they can lead to higher membrane fouling tendencies. Themore inert and cheaper polymers such as polyolefins, for example(poly(ethylene), poly(propylene) poly(vinylidene difluoride)) can beused to make microporous membranes, but these materials are veryhydrophobic. In some embodiments of the present invention, thehydrophobicity of the support member does not affect the degree offouling experienced by the composite material as the flow of liquidthrough the composite material takes place primarily in the macroporesof the gel.

In some embodiments, the porous support member is made of polymericmaterial and contains pores of average size between about 0.1 and about25 μm, and a volume porosity between 40 and 90%. Many porous substratesor membranes can be used as the support member but the support ispreferably a polymeric material, and it is more preferably a polyolefin,which, while hydrophobic, is available at low cost. Extended polyolefinmembranes made by thermally induced phase separation (TIPS), ornon-solvent induced phase separation are mentioned. Hydrophilic supportscan also be used, including natural polymers such as cellulose and itsderivatives. Examples of suitable supports include SUPOR®polyethersulfone membranes manufactured by Pall Corporation,Cole-Parmer® Teflon® membranes, Cole-Parmer® nylon membranes, celluloseester membranes manufactured by Gelman Sciences, Whatman® filter andpapers.

In some other embodiments the porous support is composed of woven ornon-woven fibrous material, for example a polyolefin such aspolypropylene. An example of a polypropylene non-woven material iscommercially available as TR2611A from Hollingsworth and Vose Company.Such fibrous woven or non-woven support members can have pore sizeslarger than the TIPS support members, in some instances up to about 75μm. The larger pores in the support member permit formation of compositematerials having larger macropores in the macroporous gel. Compositematerials with larger macropores can be used, for example, as supportson which cell growth can be carried out. Non-polymeric support memberscan also be used, such as ceramic-based supports.

The porous support member can take various shapes and sizes. In someembodiments, the support member is in the form of a membrane that has athickness of from about 10 to about 2000 μm, more preferably from 10 to1000 μm, and most preferably from 10 to 500 μm. In other embodiments,multiple porous support units can be combined, for example, by stacking.In one embodiment, a stack of porous support membranes, for example from2 to 10 membranes, can be assembled before the macroporous gel is formedwithin the void of the porous support. In another embodiment, singlesupport member units are used to form composite material membranes,which are then stacked before use.

Preparation of Composite Materials

The composite materials of the invention can be prepared by simple,single step methods. These methods can, in some instances, use water orother benign solvents, such as methanol, as the reaction solvent. Themethods also have the benefit of using rapid processes that lead toeasier and continuous manufacturing possibilities. The compositematerial is also potentially cheap.

The composite materials of the invention can be prepared, for example,by mixing one or more monomers, one or more polymers, or mixturesthereof, one or more cross-linking agents, optionally one or moreinitiators and optionally one or more porogens, in one or more suitablesolvents. The solution produced is preferably homogeneous, but aslightly heterogeneous solution can be used. The mixture is thenintroduced into a suitable porous support, where a gel forming reactiontakes place. Suitable solvents for the gel forming reaction include, forexample, water, dioxane, dimethylsulfoxide (DMSO), dimethylformamide(DMF), acetone, ethanol, N-methylpyrrolidone (NMP), tetrahydrofuran(THF), ethyl acetate, acetonitrile, toluene, xylenes, hexane,N-methylacetamide, propanol, and methanol. It is preferable to usesolvents that have a higher boiling point, as these solvents reduceflammability and facilitate manufacture. It is also preferable that thesolvents have a low toxicity, and they can be readily disposed of afteruse. An example of such a solvent is dipropyleneglycol monomethyl ether(DPM).

In some embodiments, it is possible to use dibasic esters (esters of amixture of dibasic acids) as the solvent. Dibasic esters (DBEs) areespecially suitable for preparing gels based on polyacrylamide monomers.This solvent system has an unexpected characteristic in that it ispoorly soluble in water, which differs from the other solvents usedwhich are essentially completely water miscible. While water misciblesolvents offer advantages in terms of solvent removal after fabrication,water immiscible solvents such as DBE's are good replacements, incertain cases, for solvents such as dioxane that are volatile,flammable, and toxic.

In some embodiments, components of the gel forming reaction reactspontaneously at room temperature to form the macroporous gel. In otherembodiments, the gel forming reaction must be initiated. The gel formingreaction can be initiated by any known method, for example throughthermal activation or U.V. irradiation. The reaction is more preferablyinitiated by U.V. irradiation in the presence of a photoinitiator, asthis method has been found to produce larger macropores in the gel, andit accelerates the gel forming reaction more than the thermal activationmethod. Many suitable photoinitiators can be used, of which2-hydroxy-1[4-2(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959*), and 2,2-dimethoxy-2-phenylacetophenone (DMPA) are preferred.Other suitable photoinitiators include benzophenone, benzoin and benzoinethers such as benzoin ethyl ether and benzoin methyl ether,dialkoxyacetophenones, hydroxyalkylphenones, α-hydroxymethyl benzoinsulfonic esters. Thermal activation requires the addition of a thermalinitiator. Suitable thermal initiators include1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88),azobis(isobutyronitrile) (AIBN), potassium persulfate, ammoniumpersulfate, and benzoyl peroxide.

If the reaction is to be initiated by U.V. irradiation, a photoinititoris added to the reactants of the gel forming reaction, and the supportmember containing the mixture of monomer, cross-linking agent andphotoinitiator is subjected to U.V. irradiation at wavelengths of from250 nm to 400 nm, for a period of a few seconds to a few hours. Withcertain photoinitiators, visible wavelength light may be used toinititate the polymerization. To permit the initiation, the supportmaterial must have a low absorbance at the wavelength used, to permittransmittance of the UV rays through the support. Preferably, thesupport and macroporous gel reagents are irradiated at 350 nm for a fewseconds to up to 2 hours.

Preferably, thermally initiated polymerization is carried out at 60-80°C. for a few minutes up to 16 hours.

The rate at which polymerization is carried out has an effect on thesize of the macropores obtained in the macroporous gel. As discussedearlier, when the concentration of cross-linker in a gel is increased tosufficient concentration, the constituents of the gel begin to aggregateto produce regions of high polymer density and regions with little or nopolymer, which latter regions are referred to as “macropores” in thepresent specification. It is this mechanism which is affected by therate of polymerization. When polymerization is carried out slowly, suchas when a low light intensity in the photopolymerization, theaggregation of the gel constituents has more time to take place, whichleads to larger pores in the gel. Alternatively, when the polymerizationis carried out at a high rate, such as when a high intensity lightsource is used, there is less time available for aggregation and smallerpores are produced.

Once the composite materials are prepared, they can be washed withvarious solvents to remove any unreacted components and any polymer oroligomers that are not anchored within the support. Solvents suitablefor the washing of the composite material include water, acetone,methanol, ethanol, and DMF.

Uses of the Composite Material

The composite material of the invention can find use in many differentapplications, where a liquid is passed through the macropores of thegel. The liquid passed through the macropores can be selected from, forexample, a solution or a suspension, such as a suspension of cells or asuspension of aggregates.

In some embodiments, the composite materials can be used to performseparations. Uses include size exclusion separations such asultrafiltration and microfiltration systems. The composite material ofthe invention is advantageous in these types of applications because ofthe wide range of pore-sizes available, and the ease with whichcomposite materials with different pore-sizes can be made. In someembodiments, it is preferred that the composite materials used forsize-exclusion separations are not fully occupied, i.e. that while allor substantially all the liquid that flows through the compositematerial flows through the macroporous gel, the void volume of thesupport member is not completely occupied by the macroporous gel.Composite materials having a non-fully occupied void volume where thedensity of the macroporous gel is greater at or adjacent to a firstmajor surface of the support member than the density at or adjacent to asecond major surface of the support member are referred to as beingasymmetric.

In those embodiments where the macroporous gel bears a charge, thecombination of macropore surface charge and of controlled macropore sizeproduces a composite material that can be used in Donnan type exclusionseparations. In these instances composite materials are produced thathave high charge densities coupled with high hydraulic flows (flux). Thehigh charge densities, coupled with high permeability, can be useful,for example, in adsorption of metal ions by giving composite materialswith high ion-exchange capacities. The examples will demonstrate thatthese composite materials can also be used for the recovery of proteinsand other related molecules, and that the composite materials of theinvention exhibit high binding capacities.

Composite materials of the invention are also suitable for theseparation of biomolecules, such as proteins, from solution, as thebiomolecules may have specific interactions with ligands or bindingsites found in the macropores of the composite materials. The specificinteractions may involve electrostatic interactions, affinityinteractions or hydrophobic interactions. Examples of molecules or ions,including biological molecules or ions, that can be separated includeproteins such as albumins, e.g., bovine serum albumin, and lysozyme, butthey can also be used in the separation of supramolecular assembliessuch as viruses and cells. Examples of other biomolecules that can beseparated include y-globulins of human and animal origins,immunoglobulins such as IgG, IgM, or IgE of both human and animalorigins, proteins of recombinant or natural origin including protein A,polypeptides of synthetic or natural origin, interleukin-2 and itsreceptor, enzymes such as phosphatase, dehydrogenase, etc., monoclonalantibodies, trypsin and its inhibitor, albumins of different origins,e.g., human serum albumin, chicken egg albumin, etc., cytochrome C,immunoglobulins, myoglobulin, recombinant human interleukin, recombinantfusion protein, nucleic acid derived products, DNA and RNA of eithersynthetic or natural origin, and natural products including smallmolecules. Biomolecule separations occur mostly in the macropores of thegel, but they can also take place, albeit at a slower rate, within thegel itself.

Some composite materials can be used as a reversible adsorbent. In theseembodiments, a substance, for example a biomolecule, that is adsorbed inthe macropores or in the mesh (micropore) of the gel can be released bychanging the liquid that flows through the macroporous gel. Variationsin the gel composition can be used to control the properties of the gelin terms of uptake and release of adsorbed substances. Another advantageof the composite material of the invention is that it can be made in theform of a membrane, and membrane-based biomolecule recovery is easier toscale up, less labor intensive, more rapid, and has lower capital coststhan the commonly used conventional packed column chromatographytechniques.

Some composite materials can also be used as solid supports for chemicalsynthesis or for cell growth. The reactants or nutrients required forthese processes can either continuously flow through the macropores ofthe composite material, or they can be left to reside inside themacropores and then pushed out at a later time. In one embodiment,composite materials can be used in the stepwise production of peptides.In such an application, amino acids are attached to the macroporesurface, and amino acid solutions are sequentially passed through themacropores to prepare peptide chains. The formed peptide can then becleaved from the support by passing a suitable solvent through themacropores. Supports currently used to carry out this type of synthesisconsist of beads with small pores that offer slow diffusioncharacteristics. The composite materials of the invention have a moreuniform pore structure, and they have through pores wherein the flow ofliquids is not controlled diffusion.

Application areas include but are not limited to pharmaceuticals,including biotechnology, food, beverage, fine chemicals, and therecovery of metal ions.

Uses of Responsive Composite Materials

The composite materials that comprise responsive macroporous gels(responsive composite materials) are preferably used to fractionatefluid components based on a size-exclusion mechanism. By this mechanism,steric hindrance is exerted on the convection and diffusion of amolecule or particle approaching the pores of a filtering device. Theimpediment to the transport is related to the ratio of the pore radiusin the filter to the radius of the molecule or particle. When this ratioapproaches to 1:1, molecules or particles will be completely retained bythe filtering device. By changing the pore radius, molecules orparticles of different sizes are permitted to pass through the pore andfractionation by size is achieved.

The responsive composite materials are well adapted to size exclusionmechanisms as the pores of these materials have a narrow pore-sizedistribution. This means that size exclusion separations can be achievedfor molecules that have molecular weights that are much closer thannormally permitted by conventional ultrafiltration/microfiltrationmembranes. In some embodiments, the size difference between themolecules being separated by the responsive composite materials of theinvention can be as low as about 0.9 nm. In other embodiments, theresolution is as low as about 0.5 nm. While some of the knownultrafiltration membranes do have narrow pore-size distributions (e.g.track etched membranes), these membranes have the disadvantage of beingcostly and of having relatively low fluxes.

In those embodiments where the composite materials are hydrophilic, thecomposite materials are well adapted to the size-separation of proteinsas there is little or no adsorption of molecules to the compositematerial. Normally, proteins are prone to non-specific binding. Suchresponsive composite materials are thus suitable for the separation of amulti-component protein mixture into discrete fractions based only onthe size of the protein fractions. This separation of proteins throughan exclusively size-exclusion method differs from the methods known inthe art for separating proteins, where differences in size are used inconjunction with other physicochemical effects such as electrostaticcharge, electrostatic double layer, and hydrophobic interaction effects,which physiochemical effects can lead to non-specific binding or evendenaturation of the protein. Use of the composite material to separateproteins on a size-exclusion basis thus avoids the denaturation of theprotein or its loss due to irreversible binding. The separation ofproteins with the responsive composite material of the invention is avery gentle process that does not involve strong interactions betweenthe protein and the separation medium, which permits the recovery ofproteins with higher overall efficiencies, a major factor in theeconomics of therapeutic protein recovery.

One example of separations using the responsive composite materials isthe separation of human serum albumin (HSA) (size of 60 kDa) from humanimmunoglobulin G (IgG) (size of 160 kDa). Size based separations ofproteins are possible at fixed environmental conditions. With theresponsive macroporous gel in a swollen or partially swollen state,e.g., at a low salt concentration in the feed with gels having weak acidor base functionalities, the protein with the lower molecular weight(e.g., human serum albumin) can be freely transmitted through thecomposite material, while the protein with the higher molecular weight(e.g., human immunoglobulin G) can be retained. When operated at a fixedenvironmental condition, the ultrafiltration process generates twoproduct streams, and the fixed environment mode is suitable forfractionating binary mixtures, i.e. one protein from another.

The responsive composite materials of the invention can also be used togenerate more than two product streams through size-based separationsutilising the dynamic pore-size capabilities of the responsive compositematerial. This multi-component separation is possible as the change inmembrane pore-size in response to change in environmental condition isgradual. When operated in this mode (i.e. with change in environmentalcondition), the process is suitable for separating proteins from amulti-protein mixture. In such a process, the environment is changedeither in a stepwise fashion or gradually by appropriately altering theenvironment of the responsive gel. For example, changing the pH of thebulk medium used to carry out the filtration process using binary orternary buffer systems can bring about a change in pore-size, and hencea sequential size based separation. When operated in the step changemode, each step will generate a fraction, each fraction containingproteins smaller than in the next fraction. If n number of fractions aregenerated, (n−1) of these will be obtained in the permeate and then^(th) fraction will be in the retentate.

The multi-component separations presented above represent a completelynew use of ultra- and nano-filtration membranes. This type ofmulti-component separation is also referred to as chromatographicfiltration. Some specific applications of this new type of separationinclude:

(a) fractionation of hen egg white components;

-   -   (i) LMW compounds such as Avidin (MW<1000);    -   (ii) Lysozyme (MW 14,100);    -   (iii) Ovalbumin (MW 47,000);    -   (iv) Conalbumin (MW 80,000);        (b) fractionation of human plasma proteins;    -   (i) Human serum albumin (HSA, MW 67,000);    -   (ii) Human immunoglobulin G (HIgG, MW 155,00);    -   (iv) Other human immunoglobulins (e.g. HIgM, MW>300,000);        (c) fractionation of dextran into molecular weight based        fractions;        (d) fractionation of PEG into molecular weight based fractions;        (e) fractionation of polymers into molecular weight based        fractions; and        (f) fractionation of micron-sized particles into size based        fractions.

While the composite materials of the present invention are quitesuitable for the separation of bulk materials, they can also be used toseparate components on a smaller volume scale. For example, theresponsive composite material can be used to separate biochemicalsubstances such as antibodies, other bioactive proteins, hormones,polysaccharides and nucleic acids, prior to analysis. Many of thecurrently used biospecific analytical methods, e.g. Enzyme Linked ImmunoSorbent Assay (ELISA) are based on the binding of the above substances,or the binding of substances which biospecifically interact with thesesubstances (e.g. antibodies, antigens, ligands, and substrateanalogues), onto solid surfaces such as polystyrene (as in ELISA) oronto synthetic membranes (as in immuno-blotting). The detection limitsof these tests are frequently limited by the surface area available indevices such as microwell plates or blotted sections of flat sheetmembranes. Another limitation imposed by attachment of material ontosolid surfaces is the likelihood of steric hindrance affecting thebiospecific recognitions on which these tests are based. Biospecificanalytical methods which rely on solution phase recognition and bindingare also available, e.g. Radio Immuno Assay (RIA). These methodsfrequently rely on the use of porous synthetic membranes for retainingand enriching substances which are to be analyzed. However, the fixednature of the permeability of these membranes could prove to be alimiting factor. The use of responsive composite materials wouldfacilitate sequential removal of substance from solutions containingsubstances to be analyzed and thus facilitate analysis which would notbe feasible with fixed permeability membranes. The responsive membranescan also be utilised to facilitate removal from test solutions ofsubstances which are likely to interfere with the assays.

While the responsive composite materials are especially suitable for usein size-exclusion separation, they can nonetheless be used in Donnantype separations and specific binding separations by the incorporationof appropriate monomers or polymers in the macroporous gel.

The responsiveness of composite material also permits the ability toopen the pores of a membrane (made from the composite material) afteruse, and to then return and readjust the pores to their initial valuesby reversing the environmental change. Opening of the pores facilitatesthe cleaning of these membranes by removing a fouling material, therebyprolonging the effective use of the membrane.

EXAMPLES

The following examples are provided to illustrate the invention. It willbe understood, however, that the specific details given in each examplehave been selected for purpose of illustration and are not to beconstrued as limiting the scope of the invention. Generally, theexperiments were conducted under similar conditions unless noted.

Experimental Materials Used

The monomers used were acrylamide (AAM),2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS),(3-acrylamidopropane)trimethylammonium chloride (APTAC),diallyldimethylammonium chloride (DADMAC), ethylene glycoldimethacrylatecrylate (EDMA), glycidyl methacrylate (GMA),N,N′-methylenebisacrylamide (BIS), methacrylic acid (MAA), acrylic acid(AA), and trimethylolpropane triacrylate (TRIM). The polymers used werebranched poly(ethylene imine) (BPEI) of an average molecular weight (MW)of 25000 Da, poly(ethylene glycol) (PEG) of average molecular weight of200, 1000, 2000, 4000 and 10000 Da, and poly(allylammoniumhydrochloride) (PAH) of an average molecular weight of 60000 Da. Thecross-linker used for BPEI was ethylene glycol diglycidyl ether (EDGE).

The solvents used were cyclohexanol (CHX), methylene chloride (CH₂Cl₂),deionized water, 1,4-dioxane, N,N-dimethylformamide (DMF), dodecanol(DDC), glycerol, methanol, 1-octanol, and 1-propanol.

The free radical polymerization initiators used were2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959), 2,2-dimethoxy-2-phenylacetophenone (DMPA), and1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88).

Proteins used were bovine serum albumin (BSA), lysozyme, human serumalbumin (HSA) and human immunoglobulin (HIgG). Other chemicals used wereacryloyl chloride, hydrochloric acid, sodium azide, sodium chloride,sodium hydroxide, triethylamine, tris(hydroxymethyl)aminomethane (TRIS),4-morpholineethanesulfonic acid (MES) and buffers (Tris Buffer).

The porous supports used were poly(propylene) thermally induced phaseseparation (TIPS) membranes PP1545-4 with an average pore diameter of0.45 μm, thickness of 125 μm, and porosity of 85 vol-%, produced by 3MCompany, and PP1183-3X of an average pore diameter of 0.9 μm, thicknessof 87 μm, and porosity of 84 vol-%, both produced by 3M Company, andnon-woven meltblown poly(propylene) TR2611A of a mean pore flow diameterof 6.5 μm, thickness of 250 μm, and porosity of 89.5 vol-% produced byHollingworth & Vose Company.

Preparation of Composite Materials

The composite materials of the invention can be prepared according tothe following general procedure. A weighed support member was placed ona poly(ethylene terephthalate) (PET) or poly(ethylene) (PE) sheet and amonomer or polymer solution was applied the sample. The sample wassubsequently covered with another PET or PE sheet and a rubber rollerwas run over the sandwich to remove excess solution. In situ gelformation in the sample was induced by polymerization initiated byirradiation with the wavelength of 350 nm for the period of 10 to 120minutes or by heating the sandwich at 60-80° C. for 2 hours. Theirradiation was typically carried out using a system containing four 12″long lamps, approx. 1.5″ spaced and emitting light at 365 nm with theoutput energy of approx. 0.1 Watt/inch. The system was equipped with asmall fan to dissipate the heat (no other temperature control). Theirradiated sample was located at approx. 5″ distance from the lamps. Incase when preformed polymer and in situ cross-linking was used to formthe gel, the sandwich was left at room temperature until thecross-linking reaction was completed, typically for 2-16 hours. Theresulting composite material was thoroughly washed with a suitablesolvent or a sequence of solvents and stored in a 0.1 wt-% aqueoussolution of sodium azide to prevent bacterial growth. In order todetermine the amount of gel formed in the support, the sample was driedin vacuum at room temperature to a constant mass. The mass gain due togel incorporation was calculated as a ratio of an add on mass of the drygel to the initial mass of the porous support.

Flux Measurements

Water flux measurements through the composite materials were carried outafter the samples had been washed with water. As a standard procedure, asample in the form of a disk of diameter 7.8 cm was mounted on asintered grid of 3-5 mm thickness and assembled into a cell suppliedwith compressed nitrogen at a controlled pressure. The cell was filledwith deionized water or another feed solution and a desired pressure wasapplied. The water that passed through the composite material in aspecified time was collected in a pre-weighed container and weighed. Allexperiments were carried out at room temperature and at atmosphericpressure at the permeate outlet. Each measurement was repeated three ormore times to achieve a reproducibility of ±5%.

The water flux, Q_(H2O) (kg/m²h), was calculated from the followingrelationship:

$Q_{H_{2}O} = \frac{\left( {m_{1} - m_{2}} \right)}{A \cdot t}$

where m¹ is the mass of container with the water sample, m₂ is the massof container, A is the active membrane surface area (38.5 cm²) and t isthe time.

The composite material of the invention may have water flux values thatare smaller than those of the unfilled support member, with possibleflux reduction of about a factor of two to about of a factor of a fewhundred depending on the application. For ultrafiltration application,the flux may be reduced by a factor of about ten to about a few hundred.

The hydrodynamic Darcy permeability, k (m²) of the membrane wascalculated from the following equation

$k = \frac{Q_{H_{2}O}\eta \; \delta}{3600\; d_{H_{2}O}\Delta \; P}$

where η is the water viscosity (Pa·s), δ is the membrane thickness (m),d_(H2O) is the water density (kg/m³), and ΔP (Pa) is the pressuredifference at which the flux, Q_(H2O), was measured.

The hydrodynamic Darcy permeability of the membrane was used to estimatean average hydrodynamic radius of the pores in the porous gel. Thehydrodynamic radius, r_(h), is defined as the ratio of the pore volumeto the pore wetted surface area and can be obtained from theCarman-Kozeny equation given in the book by J. Happel and H. Brenner,Low Reynolds Number Hydrodynamics, Noordhof Int. Publ., Leyden, 1973, p.393:

$k = \frac{ɛ\; r_{h}^{2}}{K}$

where K is the Kozeny constant and ε is the membrane porosity. TheKozeny constant K≈5 for porosity 0.5<ε<0.7. The porosity of the membranewas estimated from porosity of the support by subtracting the volume ofthe gel polymer.

Protein Adsorption/Desorption Experiment

Protein adsorption experiments were carried out with two proteins,namely, bovine serum albumin (BSA) and lysozyme. In the case ofexperiments with a positively charged composite material in the form ofa membrane, the membrane sample was first washed with distilled waterand subsequently with a TRIS-buffer solution (pH=7.8). In an adsorptionstep, a composite material sample in a form of a single membrane disk ofdiameter 7.8 cm was mounted on a sintered grid of 3-5 mm thickness in acell used for water flux measurements and described above. A BSAsolution, comprising from 0.4 to 0.5 mg BSA per ml of buffer solution,was poured to the cell to give a 5 cm head over the composite material.This hydrostatic pressure of 5 cm was kept constant by further additionsof the BSA solution. In a modification of this method, the cell waspressurised with compressed nitrogen. The flow rate was measured byweighing the amount of permeate as a function of time. Typical valuesvaried between 1 and 5 ml/min. Permeate samples were collected at 2-5min intervals and analyzed by UV analysis at 280 nm. Following theadsorption step, the composite material in the cell was washed withabout 200 ml of the TRIS-buffer solution, and desorption was carried outwith a TRIS-buffer solution containing 1M NaCl at 5 cm head pressure orunder a controlled pressure of compressed nitrogen. The permeate sampleswere collected at 2-5 min intervals and tested by UV analysis at 280 nmfor BSA content.

For negatively charged composite materials, a solution of lysozyme in aMES buffer solution having a pH of 5.5 and a lysozyme concentration of0.5 g/L was used in a procedure similar to that described above for BSAand positively charged materials. The flow rate during the proteinadsorption was again kept within 1-5 ml/min. Prior to the desorption ofthe protein, the membrane was washed by passing with 200 ml of thebuffer solution. The desorption of the protein was carried out using aMES buffer solution (pH=5.5) containing 1M NaCl in the same way asdescribed above for the desorption of BSA. The lysozyme content in thecollected samples was determined by UV spectrophotometry at 280 nm.

In other examples, protein adsorption tests involve stacks of severalmembranes of diameter of 19 mm mounted into a Mustang® Coin Devicemanufactured by Pall Corporation and the protein solution was deliveredto the membrane stack at controlled flow rate using a peristaltic pump.The permeate fractions were collected and analyzed in the same way asdescribed above. The desorption of the proteins was carried in a similarway as described above, with buffered 1M NaCl delivered to the membranestack by using the pump instead of gravity or compressed nitrogenpressure.

Protein Separation Experiment

The experimental method used to examine the separation properties of theresponsive composite materials of this invention in protein-proteinfractionation processes is based on the pulsed injection ultrafiltrationtechnique and its derivatives developed by Ghosh and his co-workers anddescribed in the following articles: R. Ghosh and Z. F. Cui, Analysis ofprotein transport and polarization through membranes using pulsed sampleinjection technique, Journal of Membrane Science, vol. 175, no. 1 (2000)p. 75-84; R. Ghosh, Fractionation of biological macromolecules usingcarrier phase ultrafiltration, Biotechnology and Bioengineering, vol.74, no. 1 (2001) p. 1-11; and R. Ghosh, Y. Wan, Z. F. Cui and G. Hale,Parameter scanning ultrafiltration: rapid optimisation of proteinseparation, Biotechnology and Bioengineering, vol. 81 (2003) p. 673-682and incorporated herein by reference. The experimental set-up used wassimilar to that used for parameter scanning ultrafiltration as describedin article by R. Ghosh, Y. Wan, Z. F. Cui and G. Hale, Parameterscanning ultrafiltration: rapid optimisation of protein separation,Biotechnology and Bioengineering, vol. 81 (2003) p. 673-682.

A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the responsive compositematerial experiments was one with a low salt concentration (typically5-10 mM NaCl). In all these experiments the carried phase was switchedto one with a high salt concentration (typically 1 M NaCl). The changein salt concentration within the membrane module could be tracked byobserving the conductivity of the permeate stream. The change intransmembrane pressure gave an idea about the change in membranehydraulic permeability with change in salt concentration.

Example 1

This example illustrates the formation of an unsupported porous gel,which can be used as the macroporous gel to prepare the compositematerial of the invention.

A solution containing 3.33 g of (3-acrylamidopropane)trimethylammoniumchloride (APTAC) monomer as a 75% aqueous solution, 0.373 g of N,N′-methylenebisacrylamide (BIS) cross-linker, and 0.0325 g ofIrgacure®2959 photoinitiator dissolved in 25 ml of adioxane:dimethylformamide:water mixture, with the solvent volume ratioof 71:12:17, respectively, was prepared. In this solvent mixture,dioxane is a poor solvent while DMF and water are good solvents. A totalmonomer concentration (APTAC and BIS) of 0.58 mol/L was thus obtained.The cross-linking degree was 20 mol %, based on APTAC. 5 ml of thissolution was placed in a glass vial and subjected to UV irradiation at350 nm for 2 hrs. A white gel was formed which was washed thoroughlywith de-ionized water to exchange the reaction solvent and remove theunreacted monomer or soluble oligomers.

The gel formed was mechanically very weak. A sample of the gel wasexamined using an environmental scanning electron microscope (ESEM) withwater vapor present in the sample chamber to prevent drying of the gel.The micrograph, shown in FIG. 1, has dark, cavernous areas that indicatethat a macroporous gel was formed.

Example 2

This example illustrates a method of preparing a positively chargedcomposite material of the present invention using the monomer solutionof composition described in example 1 applied to a sample of thepoly(propylene) porous support PP1545-4. The composite material wasprepared according to the general procedure described above using UVirradiation at 350 nm for 2 hours. After polymerization, the compositematerial was washed with de-ionized water for 48 hrs.

Mass gain of the resulting composite material after drying was 107 wt %,water flux was 1643±5 kg/m²h at 50 kPa and Darcy permeability was9.53×10⁻¹⁶ m².

The morphology of the gel-incorporated composite material was examinedusing ESEM in the same manner as described in Example 1. The ESEMmicrograph shown in FIG. 2 shows that the macroporous gel has beenincorporated into the host membrane. The micrograph shows a similarstructure to that of the unsupported macroporous gel shown in FIG. 1 andlittle evidence of the microporous support member.

Example 3

This example illustrates a method of preparing a negatively chargedcomposite material of the present invention, with a weak acidfunctionality.

5.50 g of vacuum-distilled methacrylic acid (MAA) monomer, 0.4925 g ofN,N′-methylenebisacrylamide cross-linker and 0.1503 g of Irgacure® 2959photoinitiator were dissolved in 25 ml of a dioxane:DMF solvent mixturewith a volume ratio of 9:1, respectively, to prepare the startingmonomer solution. The composite material was prepared using thepoly(propylene) PP1545-4 support and the general procedure for thephotoinitiated polymerization described above. The irradiation time usedwas 2 hours and the resulting membrane was washed with DMF for 24 hrsfollowed by a 48 hr wash with deionized water. The mass gain of theresulting dried membrane was 231 wt %, water flux was 4276±40 kg/m²h at50 kPa and Darcy permeability was 2.64×10⁻¹⁵ m².

The protein (lysozyme) absorption/desorption characteristics of thecomposite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 3. It can be seen that even with the singlemembrane disk, a relatively steep break through curve is obtainedindicating a uniform and narrow pore size distribution in the membrane.The composite material has a breakthrough lysozyme binding capacity of42.8 mg/mL. A desorption experiment with a buffer solution containing 1MNaCl indicated that the recovery of protein was 83.4%.

Example 4

This example illustrates the effect of the total monomer concentrationand solvent mixture on the hydraulic flow rate (flux) of compositemembranes with weak acid functionality of the type described in Example3.

A series of composite membranes (MAA1 through MAA5) were prepared usingmonomer solutions of chemical compositions listed in Table 1 and theporous support PP1545-4. The preparation procedure described in Example3 was employed.

TABLE 1 The effect of the total monomer concentration and solventmixture on water flux of composite membranes Total Monomer ConcentrationCross-linking Solvent Mixture Mass Flux at (MAA + BIS) Degree (volumepart) Gain 50 kPa Sample I.D. (mol/L) (mol-%) Dioxane DMF (wt %) (kg/m²· h) MAA1 1.71 5 8 2 71 12.2 ± 0.1 MAA2 2.19 5 8 2 153  94 ± 14 MAA32.68 5 8 2 177 1265 ± 111 MAA4 3.66 5 8 2 300 1800 ± 9  MAA5 2.68 5 9 1231 4276 ± 40 

As can be seen from Table 1, the hydraulic flow rate (flux) of compositemembranes of the present invention can be tuned by adjusting the monomerloading in the solution. Contrary to the typical trends found withhomogeneous gels, for which an increase in gel density is followed bydecrease in permeability, the increase in the mass gain in the membranesof this series results in the flux increase. Further increase in flux isachieved when the concentration of the poor solvent (dioxane) in thesolvent mixture is increased (compare samples MAA3 and MAA5).

Example 5

This example illustrates a method of preparing a negatively chargedcomposite material of the present invention that has strong acidfunctionality.

A solution containing 2.50 g 2-acrylamido-2-methyl-1-propanesulfonicacid (AMPS) monomer, 0.372 g N,N′-methylenebisacrylamide cross-linkerand 0.0353 g Irgacure® 2959 photo-initiator, dissolved in 25 ml of adioxane:H₂O mixture with a volume ratio 9:1, respectively, was used. Acomposite material was prepared from the solution and the supportPP1545-4 using the photoinitiated polymerization according to thegeneral procedure describe above. The irradiation time used was 1 hourat 350 nm. After polymerization, the membrane was extracted withde-ionized water for 48 hrs. The mass gain of the resulting membrane was74.0 wt %, water flux was 2559±40 kg/m²h at 50 kPa and Darcypermeability was 1.58×10⁻¹⁵ m².

Example 6

This example illustrates further the effect of the solvent mixturecomposition and the cross-linking degree on the hydraulic flow rate ofcomposite membranes with the strong acid functionality. A series ofcomposite membranes (AMPS1 through AMPS5) was prepared using chemicalcompositions listed in Table 2 following the general preparationprocedure and the irradiation conditions as in example 5.

TABLE 2 The effect of solvent mixture on water flux of the compositemembranes Total Cross-linking Solvent mixture Mass Flux at SampleMonomer Degree (volume part) gain 50 kPa I.D. Conc. (mol/L) (mol %)Dioxane DMF H₂O (wt %) (kg/m²h) AMPS1 0.48 20 5 5 0 92 3.2 ± 0.0 AMPS20.48 20 8 2 0 100 575 ± 12  AMPS3* 0.48 20 9 0 1 74 2559 ± 9   AMPS40.48 10 8 2 0 100 8.4 ± 0.0

AMPS3 is the composite membrane prepared in the previous Example.

As can be seen, a similar pattern to that described in example 4 wasobserved with regards to the relationship between solubility of polymerin the solvent and water flux of composite membranes. Comparison ofAMPS2 with AMPS 4 shows that hydraulic flow rate (flux) of a compositemembrane can also be adjusted by the degree of cross-linking.

Example 7

This example illustrates the effect of introducing a neutral co-monomerinto a negatively charged composite material of the present invention.

A solution containing 1.750 g of 2-acrylamido-2-methyl-1-propanesulfonicacid, 0.485 g of acrylamide, 0.868 g of N,N′-methylenebisacrylamidecross-linker, and 0.044 g of Irgacure® 2959 photo-initiator, dissolvedin 25 ml of a dioxane:DMF:H₂O mixture with a volume ratio 8:1:1,respectively, was prepared. A composite material was prepared from thesolution and the support PP1545-4 using the photoinitiatedpolymerization according to the general procedure describe above. Theirradiation time used was 1 hour at 350 nm. After polymerization, themembrane was extracted with de-ionized water for 48 hrs.

The mass gain of the resulting membrane was 103 wt %, water flux was7132±73 kg/m²·h at 100 kPa, and Darcy permeability was 4.40×10⁻¹⁵ m².

Example 8

This example illustrates one method of making a positively chargedcomposite material of this invention.

A 15 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) monomer and N,N′-methylenebisacrylamide (BIS)cross-linker in a molar ratio of 5:1, respectively, in a solvent mixturecontaining 37 wt-% water, 45 wt-% dioxane and 18 wt-% DMF. Thephoto-initiator Irgacure® 2959 was added in the amount of 1% withrespect to the mass of the monomers.

A composite material was prepared from the solution and the supportPP1545-4 using the photoinitiated polymerization according to thegeneral procedure describe above. The irradiation time used was 30minutes at 350 nm. The composite material was removed from between thepolyethylene sheets, washed with water and TRIS-buffer solution andstored in water for 24 hrs.

Several samples similar to that described above were prepared andaveraged to estimate the mass gain of the composite material. Thesubstrate gained 42.2% of the original weight in this treatment.

The composite material produced by this method had a water flux in therange of 2100-2300 kg/m² hr at 70 kPa and Darcy permeability of9.87×10⁻¹⁶ m².

The protein (BSA) adsorption characteristic of the composite materialwas examined using the general procedure for a single membrane diskdescribed above. The concentration of the protein used in thisexperiment was 0.4 g/L in 50 mM TRIS-buffer. The flow rate was 2-4ml/min. A plot of the concentration of BSA in the permeate vs. thepermeate volume is shown in FIG. 4. The composite material had a BSAbinding capacity of 48-51 mg/ml. The BSA desorption was found to be inthe range of 78-85%.

Example 9

This example illustrates that by adding a neutral monomer to the chargedmonomer used in Example 6 the protein binding capacity can besubstantially increased.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and acrylamide (AAM), in the ratio 80:20, in a solventmixture containing 63 wt-% dioxane, 18 wt-% water, 15 wt-% DMF, and 4wt-% dodecanol. N,N′-methylenebisacrylamide cross-linker was added tothe monomer solution to obtain 40% (mol/mol) cross-linking degree. Thephotoinitiator Irgacure® 2959 was added in the amount of 1% with respectto the total mass of monomers.

A composite material was prepared from the solution and the supportPP1545-4 using the photoinitiated polymerization according to thegeneral procedure describe above. The irradiation time used was 20minutes at 350 nm. The composite material was removed from between thepolyethylene sheets, washed with water, TRIS-buffer solution and storedin water for 24 hrs.

A similar sample to that described above was prepared and used toestimate the mass gain of the composite material. The substrate gained80% of the original weight in this treatment.

The composite material produced by this method had a water flux in therange of 250 kg/m² hr at 70 kPa and Darcy permeability was 1.09×10⁻¹⁶m².

The protein (BSA) adsorption characteristic of the composite materialwas examined using the general procedure for a single membrane diskdescribed above. The protein concentration was 0.4 g/L in a 50 mM TRISbuffer solution. The flow rate of absorption experiment was adjusted to2 4 ml/min. The composite material had a BSA binding capacity of 104mg/ml.

Example 10

This example illustrates the formation of a supported porous gelcomposite material by cross-linking of a pre-formed polymer.

Three separate solutions were prepared with the following compositions:(A) 20 g of branched poly(ethyleneimine) (BPEI) (25,000 Da) in 50 ml ofmethanol, (B) 20 g of poly(ethyleneglycol) PEG (˜10,000 Da) in 50 ml ofmethanol, and (C) ethyleneglycol diglycidyl ether (0.324 g) in 5 ml ofmethanol.

A mixture of the three solutions was prepared consisting of 2 ml of (A),3 ml of (B), and 5 ml of (C). A portion of this resulting solution wasallowed to stand in a vial overnight when a phase separation wasobserved. Examination of the morphology of the upper clear gel layerindicated that it was macroporous.

The same mixed solution was spread on a sample of poly(propylene)support PP1545-4 using the techniques described in the generalprocedure. The membrane was sandwiched between twopoly(ethyleneterephthalate) sheets and allowed to stand overnight. Thecomposite material was extracted with methanol at room temperature for24 h, and a mass gain of 95% was observed. The water flux of thecomposite material was 6194 kg/m² h at 100 kPa and Darcy permeabilitywas 4.89×10⁻¹⁰ m².

The dynamic protein absorption capacity of the composite material wasmeasured using a BSA solution (0.4 mg/mL) in the method for a singlemembrane disk described in the general section above. It had a capacityof 68 mg/ml before breakthrough.

Example 11

This example illustrates the effect of monomer mixture composition andthe polymerization conditions on the hydraulic properties of compositematerials prepared by in situ polymerization of glycidyl methacrylate(GMA) with ethylene dimethacrylate (EDMA) used as a cross-linker. Thesolvents used were dodecanol (DDC), cyclohexanol (CHX), and methanol. Aporous polypropylene support membrane PP1545-4 and two modes ofinitiation of in situ polymerization were used according to the generalprocedure described above. In the photopolymerization mode,2,2-dimethoxy-2-phenylacetophenone (DMPA) was used as a photoinitiatorwhile the thermal polymerization was initiated by1,1′-azobis(cyclohexanecarbonitrile). In both modes, the polymerizationwas carried out for 2 hours.

The polymerization conditions and properties of the composite materialscontaining porous poly(glycidyl methacrylate-co-ethylene diacrylate) arepresented in Table 3.

TABLE 3 Porous poly(glycidyl methacrylate-co-ethylene diacrylate)-filledcomposite materials Total Monomer Initiation Mode Mass DarcyHydrodynamic Membrane Concentration of Gain Permeability radius ID wt-%Solvent Polymerization wt-% m² nm AM612 43.8 DDC/CHX 9/91 Photo 276.86.96 × 10⁻¹⁶ 95.1 AM614 22.9 DDC/CHX 9/91 Photo 144.3 2.77 × 10⁻¹⁵ 148.5AM615 47.6 DDC/CHX 9/91 Thermal 237.0 1.66 × 10⁻¹⁷ 13.0 AM616 24.9DDC/CHX 9/91 Thermal 157.5 9.15 × 10⁻¹⁶ 86.4 AM619 48.6 Methanol Photo265.0 2.48 × 10⁻¹⁵ 163.8

The mass gain obtained in this series of composite materials isproportional to the total monomer concentration in the polymerizationmixture. Membranes AM612, AM615, and AM619 were prepared using highconcentration of monomers while in membranes AM614 and AM616 the monomerconcentration was cut approximately by half (Table 3).

The high values of the pure water flux measured at 100 kPa oftransmembrane pressure (Table 3) indicate that the pore-filling materialis macroporous. The pure water flux and, consequently, the hydraulicradius are affected not only by the mass gain but also by thepolymerization mode. As shown in FIG. 5, the hydraulic radius is alinear function of the mass gain with the slope depending on thepolymerization mode. The absolute value of the negative slope in thethermal polymerization is twice that of the photopolymerized compositematerials. This means that photopolymerized composite materials havelarger pores than that of the thermally polymerized ones at the samemass gains. Thus, the photo-initiated polymerization, which is fasterthan the thermally-initiated one, produces larger pores. Since themonomer conversion is practically the same in both cases ofpolymerization (similar mass gains), the presence of the poly(propylene)substrate either through its hydrophobic nature or by creatingmicroscopic confinements for the polymerization affects the poreformation and the final structure of the pore-filling materials.

By changing the solvents from dodecanol/cyclohexanol 9/91 to methanol,which is cheaper and environmentally more acceptable than the othersolvents, a composite material with very high flux was obtained(membrane AM619, Table 3). The composite material was produced from theconcentrated monomer mixture and had flux comparable with that of themembrane AM614 which had a mass gain almost twice as low as that ofAM619.

This and subsequent examples illustrate a feature of some compositematerials of the invention. With the capability to change thecomposition and concentration of the monomers and solvents, there can beproduced stable composite materials with different porous structures. Asshown in Table 3, composite materials with larger pores can be made inthis way.

Example 12

This example illustrates further the effect of monomer mixturecomposition on the hydraulic properties of composite materials of thisinvention.

A series of composite materials have been prepared according to thegeneral procedure described above and containing porous poly(acrylamide)gels formed by in situ photoinitiated polymerization of acrylamide (AAM)and N,N′-methylenebisacrylamide (BIS) as a cross-linker in the pores ofa poly(propylene) support membrane. The porous support member used waspoly(propylene) TIPS membrane PP1545-4.2,2-Dimethoxy-2-phenylacetophenone (DMPA) or1-[4-(2-hydroxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959) were used as photoinitiators. Irradiation was carriedout at 350 nm for 2 hours. Composition of the pore-filling solutions andthe properties of the resulting composite materials are summarized inTable 4.

TABLE 4 Composition and properties of poly(acrylamide)-filled compositematerials Total Degree Monomer Solvent 1 Solvent 2 Darcy HydrodynamicMembrane of XL Conc. Conc. Conc. Mass Permeability Radius I.D. Wt-% wt-%Name wt-% Name wt-% Gain % m² nm AM606 18.0 13.3 Water 86.6 None 0.0111.7 9.3 × 10⁻¹⁸ 8.0 AM607 18.0 13.6 Water 67.9 Methanol 18.4 107.8 2.5× 10⁻¹⁸ 4.1 AM608 18.0 14.4 Water 71.8 Glycerol 13.7 110.4 2.1 × 10⁻¹⁸3.8 AM609 16.8 12.0 Water 51.9 Glycerol 36.0 103.3 1.6 × 10⁻¹⁸ 3.3 AM61031.8 34.5 DMF 49.1 1-Propanol 16.4 307.2 3.9 × 10⁻¹⁷ 20.0 AM611 32.018.7 DMF 60.9 1-Propanol 20.5 130.3 5.3 × 10⁻¹⁶ 62.7 AM617 32.2 34.9 DMF48.4 1-Octanol 16.7 273.3 8.6 × 10⁻¹⁷ 29.0

Membranes AM606 through AM609 have been prepared using very similarconcentration of monomers (12.0-14.4 wt-%) and a similar, relativelyhigh, degree of cross-linking (16.8-18.0 wt-% of monomers). The massgains obtained with these composite materials are also very similar. Asshown in FIG. 6, there is a linear relationship between total monomerconcentration in the pore-filling solution and the mass gain achievedafter photopolymerization.

The high degree of cross-linking in the composite material prepared fromaqueous solution without non-solvent (AM606) leads to relatively highpermeability. Surprisingly, the addition of methanol or glycerol, whichare poor solvents for linear poly(acrylamide), to water, which is a goodsolvent for the linear polymer, brings about a substantial reduction inthe Darcy permeability and the hydrodynamic radius calculated on itsbasis. The reduction in permeability is higher with glycerol than withmethanol and increases with the amount of glycerol in the solution.

The use of mixtures of poor solvents, such as N,N′-dimethylformamide and1-propanol or 1-octanol, as well as the further increase of the degreeof cross-linking and total monomer concentration have been tested inmembranes AM610, AM611, and AM617. As shown in Table 4, substantiallyhigher permeabilities and hydraulic radii are obtained with all thesecomposite materials as compared to the composite materials prepared withwater as one of the solvents. This occurred despite an increase of thetotal monomer concentration; more than double in membranes AM610 andAM617 than that in the membranes prepared with water as one of thesolvents. Changing the other solvent from 1-propanol in AM610 to1-octanol in AM617 also brings about substantial increase inpermeability and hydraulic radius.

Microscopic images of the surface of membrane AM610 are shown in FIGS. 7(AFM) and 8 (ESEM). For comparison, an ESEM image of the nascent poroussupport member is also shown in FIG. 8. Both sets of images show aporous phase-separated gel covering the member surface with nodiscernible elements of the support member.

Membrane AM611 was prepared with DMF and 1-propanol but the totalmonomer concentration was just over half that of AM610. Membrane AM611shows very high flux and the hydraulic radius three times that of AM610.The ESEM images of the surface of this membrane are presented in FIG. 9.It shows a highly porous gel structure (top image) that resembles thebulk gel formed in some spots on the membrane surface but detached fromthe membrane (bottom picture).

A comparison of surfaces of membranes AM610 and AM611 is presented inFIG. 10. The large difference in the size of the structural elements inthese two gels is clearly visible.

The composite materials prepared in this example can serve asultrafiltration membranes. It has been shown that the pore size of thecomposite material and, therefore, its separation properties, can becontrolled to achieve a wide range of values.

Example 13

This example illustrates the effect of pore size of the support memberon the hydraulic flow rate (flux) through composite materials of thisinvention.

Two polypropylene support membranes of pore size 0.45 μm and 0.9 μm,PP1545-4 and PP1183-3X, respectively, were used to produce compositematerials with the same monomer mixture containing 39.4 wt-% of glycidylmethacrylate and 9.2 wt-% of ethylene diacrylate in methanol, thushaving 48.6 wt-% of monomers and 18.9 wt-% of ethylene diacrylate(cross-linker) in the monomer mixture. The photoinitiator used was DMPAin the amount of 1.3 wt-% of monomers.

The composite materials were prepared according to the general proceduredescribed above. The irradiation time was 2 hours at 350 nm. Theresulting composite materials were washed with methanol followed bydeionized water. The composite materials were tested for water flux at100 kPa to calculate the Darcy permeability and hydraulic radius. Theresults are presented in Table 5.

TABLE 5 Hydraulic properties of composite membranes produced withsubstrates of different pore sizes Average Standard Support pore MassGain Flux at 100 kPa Hydrodynamic hydrodynamic Deviation Membrane IDsize (μm) (wt-%) (kg/m²h) radius (nm) radius (nm) (%) AM619 0.45 265.08080.9 163.8 156.2 6.9 AM620 0.90 296.8 7310.1 148.5

The data show that the hydraulic radius in both composite materials isthe same within an experimental error, proving that the compositematerials contain macroporous gels of similar structure.

Example 14

This Example illustrates the synthesis of poly(ethylene glycol) (PEG,MW's 4000, 2000, 1,000, and 200) diacrylates, which can be used ascross-linkers to prepare the composite material of the invention.

The synthesis procedure used follows that described by N. Ch.Padmavathi, P. R. Chatterji, Macromolecules, 1996, 29, 1976, which isincorporated herein by reference. 40 g of PEG 4000 was dissolved in 150ml of CH₂Cl₂ in a 250-ml round bottom flask. 2.02 g of triethylamine and3.64 g of acryloyl chloride were added dropwise to the flask separately.Initially the reaction temperature was controlled at 0° C. with an icebath for 3 hrs, and then the reaction was allowed to warm to roomtemperature and kept for 12 hrs. The reaction mixture was filtered toremove the precipitated triethylamine hydrochloride salt. The filtratethen was poured into an excess of n-hexane. The colorless product,referred to as PEG 4000 diacrylate, was obtained by filtration anddrying at room temperature.

The same procedure was used with the PEG's of other molecular weights.The molar ratios of the PEG to acryloyl chloride were kept the same asused above with PEG 4000.

Example 15

This example illustrates a further method of preparing a negativelycharged composite material that has a high adsorption capacity forlysozyme.

A solution containing 0.6 g of 2-acrylamido-2-methyl-1-propanesulfonicacid (AMPS), and 0.4 g of acrylamide (AAM) as monomers, 0.25 g ofN,N′-methylenebisacrylamide (BIS) and 11.0 g of PEG 4000 diacrylateobtained in Example 14 as cross-linkers, and 0.01 g of Iragure®2959 as aphotoinitiator was prepared in 10 ml of solvent consisting of a 80:10:10volume ratio of dioxane, dimethylformamide (DMF), and water.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the composite material was prepared according tothe general procedure, with the irradiation carried out at 350 nm for 20minutes. After polymerization, the composite material was washedthoroughly with de-ionized water for 24 hrs.

Mass gain of the resulting composite material after drying was 113.2 wt%, water flux was 366±22 kg/m²h at 100 kPa, and Darcy permeability was2.26×10⁻¹⁶.

The protein (lysozyme) absorption/desorption characteristics of thecomposite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in the permeate versus the volumeof permeate is shown in FIG. 11. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a Lysozymebinding capacity of 103.9 mg/ml. A desorption experiment indicated thatthe recovery of protein was 64.0%.

Example 16

This example illustrates preparation of a negatively charged compositematerial with the same nominal polymer composition as in Example 15 butwith much higher hydraulic flows (flux) and good lysozyme uptakecapacity.

The monomer solution was produced by dilution of the solution formulatedin Example 15 with acetone with the mass ratio of 1:1.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the composite material was prepared according tothe general procedure. The irradiation time used was 2 hours. Afterpolymerization, the composite material was washed thoroughly withde-ionized water for 24 hrs.

The mass gain of the resulting composite material after drying was 51.1wt % and water flux was 6039±111 kg/m²·h at 100 kPa giving Darcypermeability of 3.73×10⁻⁵.

The protein (lysozyme) absorption/desorption characteristics of thecomposite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 12. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a lysozymebinding capacity of 75.4 mg/ml. A desorption experiment indicated thatthe recovery of protein was 65.0%.

Example 17

This example illustrates a further preparation of a negatively chargedcomposite material that has a very high flux but lower protein bindingcapacity.

The monomer solution was produced by dilution of the solution formulatedin Example 15 with acetone with the mass ratio of 1:2.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the preparation of composite material wascarried out according to the general procedure described above. UVinitiated polymerization was carried out for 2 hours. Afterpolymerization, the composite material was washed thoroughly withde-ionized water for 24 hrs.

The mass gain of the resulting composite material after drying was 34.4wt % and water flux was 12184±305 kg/m²h at 100 kPa giving Darcypermeability of 7.52×10⁻¹⁵.

The protein (lysozyme) absorption/desorption characteristics of thecomposite material were examined using the general procedure for asingle membrane disk outlined earlier. (The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min.) Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 13. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a lysozymebinding capacity of 53.5 mg/ml. A desorption experiment indicated thatthe recovery of protein was 99.0%.

Examples 15, 16 and 17 show that it is possible to control the loadingof porous gel into the host membrane thereby controlling the water fluxat a defined pressure (100 kPa in the data given in the examples) andalso that the lysozyme uptake is related to the mass of incorporatedporous gel.

Example 18

This example illustrates preparation of a negatively charged membranethat has both good protein adsorption capacity and good flux using amacromonomer.

A monomer solution containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.25 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG 2000 macromonomer obtained in Example 14,dissolved in 10 ml of a dioxane-(DMF)-water mixture with a volume ratio80:10:10, respectively, was prepared.

A microporous poly(propylene) support member in the form of a membrane,support PP1545-4, was used together with the general procedure describedabove. The irradiation time used was 20 minutes. After polymerization,the membrane was washed thoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting membrane after drying was 108.4 wt % andwater flux was 1048±4 kg/m²h at 100 kPa giving Darcy permeability of6.47×10⁻¹⁶.

The protein (lysozyme) adsorption/desorption characteristics of themembrane were examined using the general procedure for a single membranedisk outlined earlier. A relatively steep break through curve wasobtained. The membrane had a lysozyme binding capacity of 88.7 mg/ml.The desorption experiment indicated that the recovery of protein was64.0%.

Example 19

This example in combination with example 18 above further illustratesthat the protein binding capacity and flow characteristics of a membranecan be tuned.

The monomer solution was produced by dilution of the solution formulatedin Example 18 with acetone with the mass ratio of 1:1.

A porous poly(propylene) support member in the form of a membrane,support PP1545-4, was used along with the general procedure for thepreparation of composite materials described above. The irradiation timeused was 90 minutes. After polymerization, the membrane was washedthoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting membrane after drying was 45.7 wt % andwater flux was 7319±180 kg/m²h at 100 kPa.

The protein (lysozyme) absorption/desorption characteristics of themembrane were examined using the general procedure for a single membranedisk outlined earlier. A relatively steep break through curve wasobserved. The membrane had a lysozyme binding capacity of 63.4 mg/ml.The desorption experiment indicated that the recovery of protein was79.3%.

Example 20

This example illustrates the effect of a neutral co-monomer on theprotein binding capacity of composite materials of this invention.

TABLE 6 Chemical composition of stock solutions (amount of monomers in10 mL of a solution) Solvents (Dioxane/ DMF/H₂O) Stock AAM AMPSPEG2000XL BIS Irgacure ® volumetric ID (g) (g) (g) (g) 2959, (g) ratioS1 0.60 0.40 1.00 0.25 0.01 8:1:1 S2 0.40 0.60 1.00 0.25 0.01 8:1:1 S30.20 0.80 1.00 0.25 0.01 8:1:1 S4 0 1.00 1.00 0.25 0.01 8:1:1 PEG2000XL:PEG2000 diacrylate prepared in example 14

Monomer solutions were prepared by dilution of stock solutions S1-S4 inTable 6 with acetone with the mass ratio of 1:1.

Composite membranes M1-M4 were prepared by using the correspondingdiluted solutions of stocks S1-S4 and following the general preparationprocedure described earlier. The porous support used was PP1545-4 andthe irradiation time was 90 minutes. Upon completion of polymerization,the composite membranes were washed with de-ionized water for 24 hrs.

The properties and protein binding capacities of composite membraneswere examined and the results shown in Table 7. It is evident that thecharge density of polyelectrolyte gels influences significantly proteinadsorption onto membranes.

TABLE 7 Properties and Lysozyme adsorption capacities of compositemembranes Flux at Binding 100 kPa Capacity No. (kg/m² · h) (mg/ml) M18146 ± 96 56.9 M2 4273 ± 46 76.3 M3  7940 ± 303 41.5 M4 8651 ± 72 16.4

Example 21

This example illustrates the effect of the chain length of thepolyfunctional macromonomers used as cross-linkers (PEG diacrylates) onprotein binding capacity of composite materials of this invention.

A series of stock solutions containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.10 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG diacrylate with different molecular weights (200,1000, 2000, 4000), obtained in Example 14, dissolved in 10 ml of adioxane-DMF-water mixture with a volume ratio 80:10:10, respectively,was prepared. The stock solutions were subsequently diluted with acetoneat the mass ratio of 1:1. A series of composite membranes were preparedfrom these solutions using poly(propylene) support PP1545-4 and byfollowing the general preparation procedure described above. Theirradiation time used was set to 90 minutes. Upon completion ofpolymerization, the composite membranes were washed with de-ionizedwater for 24 hrs.

The properties and protein binding capacities of composite membraneswere examined according to the general procedure for a single membranedisk. The results shown in Table 8 clearly indicate that the gelstructure of composite membranes has substantial effect on proteinadsorption. Possibly, it is related to the gel structure near themacropore surface, where an extremely loose structure may be formed thatcan allow protein to penetrate into the gel layer at a certain depth.Another possibility is that by using a longer chain PEG diacrylate thesurface area is increased owing to some fuzziness at the surface andthus making more adsorption site available to proteins.

TABLE 8 Properties and Lysozyme adsorption capacities of compositemembranes Binding PEG Flux at 100 kPa Capacity diacrylate (kg/m²h)(mg/ml) 200 8390 ± 218 24.4 1000 7275 ± 139 58.1 2000 4273 ± 46  76.34000 6039 ± 111 75.4

Example 22

This example illustrates the use of fibrous non-woven support to producea composite material of this invention containing positively chargedmacroporous gel.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and acrylamide (AAM), which were taken in the ratio80:20, in a solvent mixture containing 65 wt-% of dioxane, 18 wt-% ofwater, and 17 wt-% of DMF. N,N′-methylenebisacrylamide (BIS) was addedto the monomer solution to obtain 40% (mol/mol) cross-linking degree.The photoinitiator Irgacure® 2959 was added in the amount of 1% withrespect to the total mass of the monomers.

A sample of the fibrous non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs. A duplicate sample was used to estimatethe mass gain of the composite material. The substrate gained 45% of theoriginal weight in this treatment.

The composite material produced by this method had a water flux in therange of 2320 kg/m² hr at 70 kPa.

The protein (BSA) adsorption characteristic of a mono-layer of thecomposite material was examined using the general procedures one for asingle membrane disk and one for a multi-membrane stack, as describedabove. The membrane stack contained 7 membrane layers of total thickness1.75 mm. In both experiments the protein concentration was 0.4 g/L in a50 mM TRIS buffer solution, and the flow rate of the protein solutionused was 3.1±0.1 ml/min, delivered by peristaltic pump. The breakthroughcapacity for BSA was 64 mg/ml in the single membrane experiment and 55±2mg/ml in the multi-membrane stack experiment.

Example 23

This example illustrates the use of a mixture of two monomers in makinga positively charged composite material of this invention.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and (3-acrylamidopropyl)trimethylamonium chloride(APTAC), in the ratio 50:50, in a solvent mixture containing 65 wt-%dioxane, 18 wt-% water and 17 wt-% DMF. N,N′-methylenebisacrylamide(BIS) was added to the monomer solution to obtain 40% (mol/mol)cross-linking degree. The photoinitiator Irgacure® 2959 was added in theamount of 1% with respect to the total mass of the monomers.

A sample of the non-woven polypropylene substrate TR2611A was placed ona polyethylene sheet and filled with the monomer solution. The substratewas subsequently covered with another polyethylene sheet and theresulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs.

The composite material produced by this method had a water flux in therange of 2550 kg/m² hr at 70 kPa. The mass gain determined with aduplicate sample was found to be 45%.

The protein (BSA) adsorption characteristic of the mono-layer compositematerial was examined using the general procedure for a single membranedisk described above. A solution of BSA concentration of 0.4 g/L in a 50mM TRIS buffer solution was delivered to the membrane at a flow rate of2-4 ml/min. The breakthrough capacity of the composite material was 40mg/ml.

Example 24

This example illustrates the effect of addition of a neutral monomer tothe mixture of charged monomers used in example 23.

A 15 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC), (3-acrylamido-propyl)trimethylammonium chloride(APTAC), and acrylamide (AAM), which were taken in the ratio 40:40:20,in a solvent mixture containing of 65 wt-% dioxane, 17 wt-% of water,and 18 wt-% of DMF. N,N′-methylenebisacrylamide (BIS) was added to themonomer solution to obtain 20% (mol/mol) cross-linking degree. Thephotoinitiator Irgacure® 2959 was added in the amount of 1% with respectto the total mass of the monomers.

A sample of the non-woven polypropylene substrate TR2611A was placed ona polyethylene sheet and filled with the monomer solution. The substratewas subsequently covered with another polyethylene sheet and theresulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs. The composite material produced by thismethod had a water flux of 550 kg/m² hr at 70 kPa and a mass gain(determined using a duplicate samples) of 65 wt-%.

The protein (BSA) adsorption characteristic of the mono-layer compositematerial was examined using the general procedure for a single membranedisk described above. A solution of BSA concentration of 0.4 g/L in a 50mM TRIS buffer solution was delivered to the membrane at a flow rate of3.5-4 ml/min. The breakthrough capacity of the composite material was130 mg/ml.

Example 25

This example illustrates the formation of unsupported positively chargedmacroporous gel by cross-linking of a preformed polymer.

A 10% solution of poly(allylamine hydrochloride) PAH was prepared bydissolving the polymer in a solvent mixture containing 60% water and 40%iso-propanol (2-propanol). The polymer was partially deprotonated (40%)by adding 6.67 N NaOH. Ethylene glycol diglycidyl ether (EDGE) was addedto this solution to obtain 40% (mol/mol) degree of cross-linking. Thesolution was kept at room temperature for 3 hours for gel formation bythe cross-linking reaction between the amine groups of PAH and epoxygroups of EDGE. After 3 hours, the gel was placed in a water bath forall un-reacted chemicals to leach out.

A sample of the wet gel was examined using ESEM. The micrograph shown inFIG. 14 indicates that a macroporous gel was formed with the porediameter of about 70-80 μm. The wet gel was mechanically very weak.

Example 26

This example illustrates the making macroporous gel incorporated in anon-woven fabric support.

A 10 wt-% solution of poly(allylamine hydrochloride) (PAH) was preparedas in Example 25. The polymer was partially deprotonated and EDGE addedas described in Example 25 and the solution was applied to a sample ofthe non-woven polypropylene membrane support TR2611A placed between twopolyethylene sheets. The resulting sandwich was run between two rubberrollers to press the polymer solution into the pores of the substrate,spread it evenly, and remove the excess solution. The solution-filledsupport sample was kept at room temperature for 3 hours forcross-linking process to take place resulting in the formation of gel.After that time, the composite material was removed from the sandwichand placed in a water bath for 12 hours to leach out unreactedchemicals.

A wet sample of the resulting composite membrane was examined usingESEM. The micrograph shown in FIG. 15 indicates the composite membranehaving macroporous gel in the fibrous non-woven support member. Theaverage pore size of the gel was about 25-30 μm. The membrane thicknesswas 800 μm and the water flux measured at 100 kPa was 592 kg/m²h. Thecomposite material showed rather low BSA binding capacity of about 10mg/ml.

Example 27

This example provides a comparison of the protein adsorption by acomposite membrane of this invention with the commercial Mustang® Coin Qproduced by Pall Corporation.

A composite material prepared in example 22 was tested in amulti-membrane stack of 7 membrane layers of a total thickness of 1.75mm, according to the testing protocol described in example 22. AMustang® Coin Q was also tested under similar conditions. The membranestack was prepared by placing seven (7) layers of the membrane sample inthe wet state on top of each other. The assembled membrane stack waslightly compressed pressed to remove excess of water. The membrane stackwas then heated in an oven at 60-70° C. for at least 30 min. Thethickness of the resulting membrane stack in the dry state was 1.8-1.9mm. This process produced a stack in which the multiple membrane layersadhered to each other. The results shown in FIG. 16 indicate that bothsystems give similar performances.

Example 28

This example provides the hydrodynamic (Darcy) permeability of referencecomposite materials containing porous support member and homogeneousgels filling the pores of the support. The homogeneous gels wereobtained by using thermodynamically good solvents and their homogeneitywas assessed based on transparency of simultaneously formed unsupportedgels of the same composition. Clear and transparent gels were assumed tobe homogeneous, contrary to macroporous gels that were always foundopaque.

(A) Glycidyl Methacrylate Based Homogeneous Gel-Filled Composites

The composite materials containing homogeneous gels of glycidylmethacrylate-co-ethylene glycol dimethacrylate, GMA-co-EDMA, wereprepared using 1,4-dioxane as a solvent and 4.7 wt-% of EDMA(cross-linker) in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 and the generalprocedure for preparing the composite materials of this invention wereused. DMPA was used as photoinitiator and the irradiation was carriedout at 350 nm for 120 minutes. The hydrodynamic permeability of themembranes was measured and an empirical equation was derived for therelationship between the hydrodynamic permeability, k, and the mass gainof the composite membranes containing poly(GMA-co-EDMA) homogeneous gelsin the PP1545-4 support. The equation is as follows:

k=3.62×10³ ×G ^(−9.09)

The differences between the measured values of permeability and thatcalculated from the above equation were found to be less than ±3%. Thisempirical relationship was subsequently used to calculate permeabilityof reference composite materials at different mass gains.

(B) Poly(diallyldimethylammonium Chloride) Based Homogeneous Gel-FilledComposites

The composite materials containing homogeneous gels ofdiallyldimethylammonium chloride-co-methylenebisacrylamide,DADMAC-co-BIS, were prepared using water as a solvent and 1.0 wt-% ofBIS cross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the compositematerials of this invention was used to make homogeneous gel-filledmembranes. Irgacure® 2959 was used as photoinitiator and the irradiationwas carried out at 350 nm for 30-40 minutes. The hydrodynamicpermeability of the series of membranes was measured and an empiricalequation was derived for the relationship between the Darcypermeability, k, and the mass gain, G:

k=2.09×10⁻² ×G ^(−4.01)

(C) Acrylamide Based Homogeneous Gel-Filled Composites

The hydrodynamic permeability of homogeneouspoly(acrylamide)-co-methylenebisacrylamide, AAM-co-BIS, was estimatedfrom the empirical relationship between the gel permeability and the gelpolymer volume fraction provided by Kapur et al. in Ind. Eng. Chem.Res., vol. 35 (1996) pp. 3179-3185. According to this equation, thehydrodynamic permeability of a poly(acrylamide) gel,k_(gel)=4.35×10⁻²²×φ^(−3.34), where φ is the polymer volume fraction inthe gel. In the same article, Kapur et al. provide a relationshipbetween the hydrodynamic permeability of a gel and a porous membranefilled with the same gel. According to this relationship, thepermeability of the membrane, k_(membrane)=(ε/τ)×k_(gel), where ε is theporosity of the support and τ is the tortuosity of the support pores.The pore tortuosity can be estimated as a ratio of the Kozeny constant,K, for a given porosity, i.e., K=5, and the Kozeny constant for astraight cylindrical capillary equal to 2. Thus, for the poly(propylene)support PP1545-4 with porosity of 0.85, the ratio (ε/τ)=0.85/2.5=0.34.

The polymer volume fraction, φ, can be converted to mass gain using thepartial specific volume, ν₂, for poly(acrylamide) and the density, ρ, ofpoly(propylene). The values of these parameters can be found in PolymerHandbook, edited by Brandrup et al., Chapter VII, Wiley and Sons, NewYork, 1999. Thus, the mass gain of a composite material containingpoly(propylene) support of porosity εfilled with a gel whose polymeroccupies the fraction φ of the pores is given by

${{Mass}\mspace{14mu} {Gain}\mspace{14mu} (\%)} = {\frac{\phi/v_{2}}{\left( {1 - ɛ} \right)\rho} \times 100\%}$

The above equation was combined with that of Kapur et al. to give anempirical relationship allowing one to calculate hydrodynamicpermeability of reference composite materials, k, at different massgains, G. The combined equation is as follows:

k=1.80×10⁻¹² ×G ^(−3.34)

The equation is valid for ρ=0.91 g/cm³; ε=0.85; ν₂=0.7 cm³/g;(ε/τ)=0.34.

(D) Poly(AMPS) Based Homogeneous Gel-Filled Composites

The composite materials containing homogeneous gels of2-acrylamido-2-propane-1-sulfonic acid-co-methylenebisacrylamide,AMPS-co-BIS, were prepared using water as a solvent and 10.0 wt-% of BIScross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the compositematerials of this invention was used to make homogeneous gel-filledmembranes. Irgacure® 2959 was used as photoinitiator and the irradiationwas carried out at 350 nm for 60 minutes. The hydrodynamic permeabilityof the series of membranes was measured and an empirical equation wasderived for the relationship between the Darcy permeability, k, and themass gain, G:

k=2.23×10⁻¹⁶ ×G ^(−1.38)

(E) Poly(APTAC) Based Homogeneous Gel-Filled Composites

The composite materials containing homogeneous gels of(3-acrylamidopropane)trimethylammoniumchloride-co-methylenebisacrylamide, APTAC-co-BIS, were prepared usingwater as a solvent and 10.0 wt-% of BIS cross-linker in monomer mixture,and different total monomer concentrations. Poly(propylene) supportPP1545-4 was coated with Triton X-114 surfactant by immersing thesupport samples in 2t-% solution of the surfactant in methanol/water(60/40) mixture for 16 hours followed by drying in air. The generalprocedure for preparing the composite materials of this invention wasused to make homogeneous gel-filled membranes. Irgacure® 2959 was usedas photoinitiator and the irradiation was carried out at 350 nm for 60minutes. The hydrodynamic permeability of the series of membranes wasmeasured and an empirical equation was derived for the relationshipbetween the Darcy permeability, k, and the mass gain, G:

k=9.5×10⁻¹⁶ ×G ^(−1.73)

(F) Poly(ethyleneimine) Based Homogeneous Gel-Filled Composites

The composite materials containing homogeneous gels of branchedpoly(ethyleneimine) cross-linked with ethylene glycol diglycidyl ether(EDGE) were prepared using methanol solutions of BPEI of differentconcentrations. The degree of cross-linking used was 10 mol-%.Poly(propylene) support PP1545-4 was used together with the generalprocedure of fabrication of composite materials by in situ cross-linkingof cross-linkable polymers described in Example 10. A series ofmembranes with different mass gains was prepared by changing theconcentration of PEI in the solution. The Darcy permeability of themembranes was measured and an empirical equation describing therelationship between the permeability, k, and the mass gain, G, wasderived. The equation is as follows:

k=4.38×10¹⁴ ×G ^(−2.49)

Example 29

This example provides comparison between Darcy permeability of compositematerials of this invention containing supported macroporous gels andthe permeability of the reference composite materials containinghomogeneous gels filling the porous support member used in thisinvention.

The comparison is shown in Table 9 below.

TABLE 9 Permeability Ratio Values for Composite Materials CompositeMaterials Darcy containing Macroporous Gels Permeability of thisInvention of Reference Darcy Composite Permeability PermeabilityMaterial Ratio Example Mass k_(macroporous) k_(homogeneous)k_(macroporous)/ No. Gain % m² m² k_(homogeneous) 2 107 9.53 × 10⁻¹⁶2.93 × 10⁻¹⁹ 3.2 × 10³ 5  74 1.58 × 10⁻¹⁵ 2.49 × 10⁻¹⁸ 6.3 × 10² 6  92*1.98 × 10⁻¹⁸* 1.85 × 10⁻¹⁸* 1.1 × 10⁰* 100 3.53 × 10⁻¹⁶ 1.65 × 10⁻¹⁸ 2.2× 10²  100* 5.19 × 10⁻¹⁸* 1.65 × 10⁻¹⁸* 3.2 × 10⁰* 7 103  4.4 × 10⁻¹⁵1.58 × 10⁻¹⁸ 2.8 × 10³ 8  42 9.87 × 10⁻¹⁶ 8.81 × 10⁻¹⁹ 1.1 × 10³ 9  801.09 × 10⁻¹⁶ 6.78 × 10⁻²⁰ 1.6 × 10³ 10   95 1.89 × 10⁻¹⁵ 5.40 × 10⁻¹⁹3.5 × 10³ 11  144 2.77 × 10⁻¹⁵ 8.39 × 10⁻¹⁹ 3.3 × 10¹ 158 9.15 × 10⁻¹⁶3.77 × 10⁻¹⁷ 2.4 × 10¹ 237 1.66 × 10⁻¹⁷ 9.20 × 10⁻¹⁹ 1.8 × 10¹ 265 2.48× 10⁻¹⁵ 3.34 × 10⁻¹⁹ 7.5 × 10³ 277 6.96 × 10⁻¹⁶ 2.24 × 10⁻¹⁹ 3.1 × 10³*denotes homogeneous or micro-heterogeneous gels in composite materials(Comparative) 12  103 1.59 × 10⁻¹⁸* 3.38 × 10⁻¹⁹* 4.7 × 10⁰* 108 2.52 ×10⁻¹⁸* 2.93 × 10⁻¹⁹* 8.6 × 10⁰* 110 2.11 × 10⁻¹⁸* 2.70 × 10⁻¹⁹* 7.8 ×10⁰* 112 9.32 × 10⁻¹⁸ 2.61 × 10⁻¹⁹ 3.6 × 10¹ 130 5.34 × 10⁻¹⁶ 1.56 ×10⁻¹⁹ 3.4 × 10³ 273 8.57 × 10⁻¹⁷ 1.31 × 10⁻²⁰ 6.5 × 10³ 307 3.88 × 10⁻¹⁷8.87 × 10⁻²¹ 4.4 × 10³ 15  113 2.26 × 10⁻¹⁶ 1.39 × 10⁻¹⁸ 1.6 × 10² 16  51 3.73 × 10⁻¹⁵ 4.16 × 10⁻¹⁸ 9.0 × 10² 17   34 7.52 × 10⁻¹⁵ 7.18 ×10⁻¹⁸ 1.0 × 10³ 18  108 6.47 × 10⁻¹⁶ 1.47 × 10⁻¹⁸ 4.4 × 10² 19   46 4.52× 10⁻¹⁵ 4.85 × 10⁻¹⁸ 9.3 × 10² *denotes homogeneous ormicro-heterogeneous gels in composite materials.

Examples 30-37

These Examples illustrate a method of preparing a responsive compositematerial of the present invention using photoinitiated free radicalpolymerization of acrylic acid (AA) (ionic monomer), acrylamide (AA),and trimethylolpropane triacrylate (TRIM) as a cross-linker. The molarratio of acrylic acid to acrylamide was 1:1 and 1,4-dioxane was used asa solvent in all experiments. Monomer solution compositions andpolymerization conditions are given in Table 10. After polymerization,the responsive composite material was washed with de-ionized water forabout 16 hrs.

TABLE 10 Monomer solution compositions and polymerization conditionsTotal Concentration of Monomer Degree of Irradiation Example SupportMixture Cross-linking Time Mass Gain no. Sample ID Member (wt-%) (mol-%)(min) (%) 30 AM675 TR2611A 19.9 5.0 20 115.6 31 AM678 TR2611A 13.3 5.090 81.7 32 AM680 TR2611A 12.8 5.2 20 82.3 33 AM681 TR2611A 12.6 10.8 1588.8 34 AM682 TR2611A 23.8 10.9 15 167.3 35 AM684 TR2611A 31.0 10.8 10217.3 36 AM683 TR2611A 38.8 10.8 15 294.6 37 AM694 PP 1545-4 24.0 10.210 218.0

The amount of gel formed in the support member depends on the porevolume available to fill, the total concentration of the monomermixture, and the degree of conversion in the polymerization. In FIG. 17,the mass gain obtained with support TR2611A is plotted as a function oftotal monomer concentration. The data can be approximated to fall withina straight line (R²=0.97), indicating a similar degree of conversion foreach sample. The experimental values are very close to their theoreticalcounterparts estimated from the pore volume in the support and themonomer concentration. This suggests that the degree of conversion isclose to 100% and that an irradiation time of 10 minutes is sufficientunder the light conditions applied. As expected, the mass gain obtainedwith the PP 1545-4 support was higher than that obtained with theTR2611A support due to the larger porosity of the former (85 vol-%versus 79.5 vol-%).

Example 38

This example illustrates the responsiveness of the responsive compositematerials according to Example 30 to ionic interactions. For thispurpose, the composite materials were tested with solutions of differentpH and/or salt concentrations by measuring the flux at 100 kPa. Atypical change in flux taking place with the change of pH from about 3(1 mM HCl) to about 12 (1 mM NaOH), obtained with membrane AM675 isshown in FIG. 18. It can be seen from the Figure that the flux measuredwith 1 mM HCl is nearly 100 times larger than the flux measured with 1mM NaOH. The reason for this behavior of the membranes lies in changesin the degree of ionization of the acid component of the macroporousgel. At high pH (1 mM NaOH) the carboxyl groups of the acid componentbecome ionized and the electrostatic repulsive force causes the polymerchains to uncoil and stretch until balanced by counteracting forces ofthe polymer network elasticity and confinement imposed by the supportmember of the membrane. The swelling polymer chains reduce the porevolume and the pore radius in the gel. At low pH (1 mM HCl), thecarboxyl groups are converted into neutral carboxylic acid groups, theelectrostatic forces disappear, and the gel shrinks (collapses)enlarging the pores in the gel. The presence of the support memberprevents the gel from collapsing as a whole, i.e., from the process thatwould occur in the unsupported bulk gel, and closing the pores. Thus,the presence of the support reverses the direction in which hydraulicproperties of the gel change. When pure water flux is measured, thevalues obtained depend on the distance from equilibrium ionization atthe water pH (˜5.5). The initial water flux can be assumed to bemeasured at equilibrium. Immediately after the acid or base, the gel isfar from equilibrium with water and the pure water flux reflects thisstate by being close to the flux in the ionized form (after NaOH) orneutral form (after HCl).

The ratio of the flux measured with 1 mM HCl to that measured with 1 mMNaOH has been taken as a measure of membrane response (MR). The resultsobtained with membranes described in Example 30-37 are shown in Table11.

TABLE 11 Results for Examples 30-37 Membrane ID AM675 AM678 AM680 AM681AM682 AM684 AM683 AM694 Total 19.9 13.3 12.8 12.6 23.8 31.0 38.8 24.0Monomer Conc. Wt-% Degree 5.0 5.0 5.2 10.8 10.9 10.8 10.8 10.2 of Cross-linking Mol-% Membrane 89.6 372.2 352.0 29.4 10.3 5.6 4.8 19.5 Response(MR)

The results in Table 11 show that the response of the compositemembranes of this invention to ionic interaction can also be controlledby the total concentration of monomer mixture and the degree ofcross-linking. As the monomer concentration increases, the membranesensitivity to the environmental changes decreases. Similar effect isfound when the degree of cross-linking is increased.

Example 39

This example illustrates the ability of membranes based on responsivecomposite materials of this invention to fractionate proteins. Theseparation of therapeutic proteins Human Serum Albumin (HSA) and HumanImmunoglobulin G (HIgG) was chosen as a case study. Human plasma is thestarting material for the production of a number of therapeuticproteins, which are referred to as plasma proteins. The most abundantamongst these are HSA and HIgG, both of which are manufactured in bulkquantities. These proteins are generally fractionated by precipitationbased processes which give high product throughput but poor resolutionin terms of separation. Membrane based processes such asultrafiltrations have the potential for giving both high throughput andhigh resolution.

Two composite membranes of this invention containing responsivemacroporous gel, duplicates of membrane AM695 (see Tables 10 and 11),were tested for their suitability in separation of these plasmaproteins. In the experiments discussed here, the change in membrane poresize with change in salt concentration was utilized to effectprotein-protein separation in the manner desirable, i.e. sequentialrelease from the membrane module. Other environmental conditions such aspH could well be used to achieve a similar objective.

A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the experiments was onewith a low salt concentration (typically 5-10 mM NaCl). In all theexperiments the carried phase was switched to one with a high saltconcentration (typically 1 M NaCl). The change in salt concentrationwithin the membrane module could be tracked by observing theconductivity of the permeate stream. The change in transmembranepressure gave an idea about the change in membrane hydraulicpermeability with change in salt concentration. FIG. 19 shows thechanges of transmembrane pressure and conductivity as a function of thepermeate salt concentration (FIG. 19. A and B) and the changes oftransmembrane pressure as a function of permeate conductivity (FIG.19C). In this experiment the salt concentration was being increasedcontinuously in a linear fashion. The transmembrane pressure observed isrelated to the permeate salt concentration and reflects changes inpore-diameter.

Experiments were carried out using human serum albumin and humanimmunoglobulin mixtures. The ultrafiltration was started at a low saltconcentration (i.e. 10 mM). At this condition, human serum albumin wastransmitted while human immunoglobulin G was almost totally retained.The salt concentration was then increased and this increased the porediameter (as evident from drop in pressure in constant permeate fluxultrafiltration). This in turn led to the transmission of humanimmunoglobulin G through the membrane. Hence by altering theenvironmental condition it was possible to sequentially transmitproteins having different sizes through the same membrane. If theinitial mixture had also contained a protein significantly larger thanhuman immunoglobulin, it would have been possible to fractionate thethree proteins (i.e. human serum albumin, human immunoglobulin and thesignificantly bigger protein) by appropriately controlling the change insalt concentration. Two of the three fractions obtained here would be inthe permeate while the third fraction would be in the retentate.

The results obtained with duplicates of membrane AM694 are shown inFIGS. 20, 21, and 22. FIG. 20 shows the results obtained with HIgGultrafiltration. As evident from the figure, very little, if any HIgGwas transmitted at the low salt concentration. However, when the saltconcentration was increased, the HIgG was released from the membranemodule. The drop in TMP with increase in salt concentration was due tothe increase in pore diameter.

The results presented in FIG. 21 were obtained with HSA ultrafiltration.As evident from the figure HSA was freely transmitted through themembrane even at low salt concentration. When the salt concentration wasincreased, the transmission of HSA was found to increase a bit.

FIG. 22 shows the results obtained with HSA/HIgG ultrafiltration. At lowsalt concentration, HSA alone was transmitted. Ultrafiltration wascontinued until HSA was nearly completely removed from the membranemodule. The HIgG was then released by increasing the salt concentration.

Example 40

This example illustrates a method for making a positively chargedcomposite material of the invention having a high protein bindingcapacity.

A 15 wt-% solution was prepared by dissolving(3-acrylamidopropyl)-trimethylammonium chloride (APTAC),N-(hydroxymethyl)acrylamide and N,N′-methylenebisacrylamide ascross-linker in a molar ratio of 1:0.32:0.1, respectively, in a solventmixture containing 10 wt-% water, 60 wt-% di(propylene glycol)methylether and 30 wt-% dimethylformamide (DMF). The photo-initiator Irgacure®2959 was added in the amount of 1% with respect to the mass of themonomers.

A sample of the fibrous non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and to remove any excess solution. Thesubstrate was irradiated for 5 minutes at 350 nm. The composite materialwas then removed from between the polyethylene sheets, washed with waterand TRIS-buffer solution and stored in water for 24 hrs.

Several samples were prepared according to the above process, and thesamples were then dried and weighed. The average mass gain of thecomposite material was 55.7% of the original weight of the startingsupport member.

The protein (BSA) adsorption characteristic of a multi-membrane stack ofthe above composite material was examined using the general procedurefor a mono-layer of the composite material, as described earlier. Themembrane stack tested contained 4 membrane layers, giving a totalthickness 1.05 mm. The protein solution used was a 25 mM TRIS buffersolution with a protein concentration of 0.4 g/L, and the flow rate ofthe protein solution was 5.0 ml/min at 150 kPa. The breakthroughcapacity for BSA was 281 mg/ml. In a subsequent desorption step,approximately 85% of the BSA was recovered.

All references mentioned herein are incorporated herein by reference tothe same extent as if each reference were stated to be specificallyincorporated herein by reference.

To those skilled in the art, it is to be understood that many changes,modifications and variations could be made without departing from thespirit and scope of the present invention as claimed hereinafter.

1-72. (canceled)
 73. A composite material comprising: (a) a supportmember comprising a plurality of pores extending through the supportmember; and (b) a non-self supporting macroporous cross-linked gelcomprising macropores having an average size of 10 nm to 3000 nm, saidmacroporous gel being located in the pores of the support member;wherein said macroporous cross-linked gel is present in the pores of thesupport member in an amount sufficient such that, in use, liquid passingthrough the composite material passes through said macropores of saidmacroporous cross-linked gel; said macropores of said macroporouscross-linked gel are smaller than said pores of said support member; themacroporous cross-linked gel is a gel bearing a plurality of functionalgroups; and the functional groups are independently first biologicalmolecules or first biological ions.
 74. The composite material of claim73, wherein the first biological molecules or first biological ions areindependently selected from the group consisting of antibodies,fragments of antibodies, amino acid ligands, antigens, antibody ligands,anti-antibodies, and lectins.
 75. The composite material of claim 73,wherein the first biological molecules or first biological ions areindependently antibodies or fragments of antibodies.
 76. The compositematerial of claim 73, wherein the first biological molecules or firstbiological ions are antibodies; and the antibodies are monoclonalantibodies.
 77. The composite material of claim 73, wherein the firstbiological molecules or first biological ions are independently antigensor antibody ligands.
 78. The composite material of claim 73, wherein thefirst biological molecules or first biological ions are antibodyligands; and the antibody ligands are bacterial immunoglobulin-bindingproteins.
 79. The composite material of claim 78, wherein the bacterialimmunoglobulin-binding proteins are independently selected from thegroup consisting of protein A, protein G, and protein L.
 80. Thecomposite material of claim 78, wherein the bacterialimmunoglobulin-binding protein is recombinant protein A.
 81. Thecomposite material of claim 73, wherein the first biological moleculesor first biological ions are amino acid ligands.
 82. The compositematerial of claim 81, wherein the amino acid ligands areL-phenylalanine, tryptophan, or L-histidine.
 83. The composite materialof claim 73, wherein the first biological molecules or first biologicalions are lectins.
 84. The composite material of claim 83, wherein thelectin is jacalin.
 85. A method for separating a substance from a fluid,comprising the step of: placing the fluid in contact with a compositematerial that displays a specific interaction for the substance, whereinthe composite material comprises: (a) a support member comprising aplurality of pores extending through the support member; and (b) anon-self supporting macroporous cross-linked gel comprising macroporeshaving an average size of 10 nm to 3000 nm, said macroporous gel beinglocated in the pores of the support member; wherein said macroporouscross-linked gel is present in the pores of the support member in anamount sufficient such that, in use, liquid passing through thecomposite material passes through said macropores of said macroporouscross-linked gel; said macropores of said macroporous cross-linked gelare smaller than said pores of said support member; the macroporouscross-linked gel is a gel bearing a plurality of functional groups; andthe functional groups are independently first biological molecules orfirst biological ions; thereby adsorbing or absorbing the substance tothe composite material.
 86. The method of claim 85, wherein the specificinteraction is an electrostatic interaction.
 87. The method of claim 85,wherein the specific interaction is an affinity interaction.
 88. Themethod of claim 85, wherein the specific interaction is a hydrophobicinteraction.
 89. The method of claim 85, wherein the substance isindependently a second biological molecule or a second biological ion.90. The method of claim 89, wherein the second biological molecule orsecond biological ion is independently selected from the groupconsisting of a virus, a cell, a γ-globulin, an immunoglobulin, aprotein, a polypeptide, interleukin-2, interleukin-2 receptor, anenzyme, a monoclonal antibody, trypsin, trypsin inhibitor, cytochrome C,myoglobulin, a recombinant human interleukin, a recombinant fusionprotein, a nucleic acid-derived product, DNA, and RNA.
 91. The method ofclaim 89, wherein the second biological molecule or second biologicalion is a protein.
 92. The method of claim 91, wherein the protein isindependently selected from the group consisting of an albumin, alysozyme, and protein A.
 93. The method of claim 91, wherein the proteinis an albumin; and the albumin is independently selected from the groupconsisting of bovine serum albumin, human serum albumin, and chicken eggalbumin.
 94. The method of claim 89, wherein the second biologicalmolecule or second biological ion is an immunoglobulin.
 95. The methodof claim 94, wherein the immunoglobulin is independently selected fromthe group consisting of IgA, IgG, IgM, and IgE.
 96. The method of claim89, wherein the second biological molecule or second biological ion isan enzyme.
 97. The method of claim 96, wherein the enzyme isindependently a phosphatase or a dehydrogenase.
 98. The method of claim89, wherein the second biological molecule or second biological ion isindependently a y-globulin or an immunoglobulin; and the firstbiological molecules or first biological ions of the composite materialare amino acid ligands.
 99. The method of claim 89, wherein the secondbiological molecule or second biological ion is an immunoglobulin; andthe first biological molecules or first biological ions of the compositematerial are independently antigens or antibody ligands.
 100. The methodof claim 89, wherein the second biological molecule or second biologicalion is IgA; and the first biological molecules or first biological ionsof the composite material are lectins.
 101. The method of claim 89,wherein the second biological molecule or second biological ion is IgG;and the first biological molecules or first biological ions of thecomposite material are protein A.
 102. The method of claim 89, whereinthe second biological molecule or second biological ion is IgG; and thefirst biological molecules or first biological ions of the compositematerial are protein G.